Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor.

AIMS: To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation. METHODS: RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay. RESULTS: Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction. CONCLUSIONS: These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.

Heterogeneity in ornithine cytotoxicity of bovine retinal pigment epithelial cells in primary culture.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration caused by hyperornithinemia and a deficiency of ornithine-delta-aminotransferase (OAT). We recently showed that ornithine exhibits cytotoxicity to human retinal pigment epithelial (RPE) cell lines treated with the OAT inhibitor, 5-fluoromethylornithine (5-FMOrn), and suggested that this system may be an in vitro model of gyrate atrophy. In the present study, in order to apply this system to primary cultured RPE cells, we freshly prepared RPE cells from bovine eyes and studied the effect of ornithine on cell damage. Two phenotypes, epithelioid and fusiform, which coexisted in the primary culture and epithelioid phenotype cells, but not fusiform ones, were severely damaged and partially detached from the substrate by 10 m m ornithine and 0.5 m m 5-FMOrn. Neither ornithine nor 5-FMOrn alone exhibited such cytotoxicity to both phenotypes of RPE cells. Proline significantly prevented the ornithine-induced cytotoxicity. Epithelioid and fusiform phenotypes isolated from the primary culture showed different distribution of actin filaments. A combination of ornithine and 5-FMOrn time-dependently inhibited [(3)H]thymidine incorporation in the epithelioid, but not fusiform, cells. Proline prevented the inhibition of [(3)H]thymidine incorporation by ornithine in 5-FMOrn-treated epithelioid cells. Furthermore, l -azetidine-2-carboxylic acid, a collagen synthesis inhibitor, reduced [(3)H]thymidine incorporation in epithelioid, but not fusiform, cells, which was reversed by proline. These results demonstrate that the epithelioid phenotype of bovine RPE cells becomes susceptible to ornithine following inactivation of OAT. The phenotypic cells and its prevention by proline may provide insight into biochemical triggers that induce gyrate atrophy.

Cloning and characterization of two novel aldo-keto reductases (AKR1C12 and AKR1C13) from mouse stomach.

In contrast to hepatic hydrosteroid dehydrogenases (HSDs) of the aldo-keto reductase family (AKR1C), little is known about a stomach one. From a mouse stomach cDNA library, we isolated two clones encoding proteins of 323 amino acid residues. They exhibited 93.2% amino acid sequence identity and 64-68% with any known HSDs. Recombinant proteins expressed in Escherichia coli reduced 9,10-phenanthraquinone with NAD(P)H as cofactor. The mRNAs were exclusively expressed in stomach, liver and ileum. The present study demonstrates that these proteins are new members of the HSD subfamily and they are named AKR1C12 and AKR1C13. Immunohistochemical analysis suggests that they are involved in detoxification of xenobiotics in the stomach.

Immunosuppressant FK506 inhibits inducible nitric oxide synthase gene expression at a step of NF-kappaB activation in rat hepatocytes.

BACKGROUND/AIMS: Recent evidence indicates that an increase in nitric oxide production after liver transplantation is associated with acute allograft rejection. Nitric oxide mediates cellular injury under various pathological conditions in the liver. Studies were performed to determine whether the immunosuppressants FK506 and cyclosporin A directly influence gene expression of inducible nitric oxide synthase by interleukin 1beta in hepatocytes. METHODS: Primary cultures of rat hepatocytes were treated with interleukin 1beta in the presence and absence of FK506 or cyclosporin A. Release of nitrite (nitric oxide metabolite) into culture medium, levels of inducible nitric oxide synthase protein and mRNA, and activation of nuclear factor-kappaB were compared with the two drugs. RESULTS: Interleukin 1beta increased levels of inducible nitric oxide synthase protein and inducible nitric oxide synthase mRNA, as well as nitric oxide production, in the cultured hepatocytes. Nuclear factor-kappaB, an important transcription factor in inducible nitric oxide synthase gene expression in response to inflammation, also appeared in the nuclear fraction of hepatocytes after addition of interleukin 1beta. FK506 markedly inhibited the nitric oxide formation, inducible nitric oxide synthase protein synthesis and inducible nitric oxide synthase mRNA expression induced by interleukin 1beta, but cyclosporin A had no effects. Furthermore, FK506 inhibited nuclear factor-kappaB activation and decreased mRNA levels of the p50/p65 subunits of nuclear factor-kappaB. CONCLUSIONS: These results demonstrate that FK506, but not cyclosporin A, inhibits the induction of inducible nitric oxide synthase expression during nuclear factor-kappaB activation. FK506 may influence liver function during diseases by modulating the nitric oxide pathway, in addition to its immunosuppressive effect.

Synergistic regulation of inducible nitric oxide synthase gene by CCAAT/enhancer-binding protein beta and nuclear factor-kappaB in hepatocytes.

BACKGROUND: Nitric oxide (NO) has diverse activities under physiological and pathophysiological conditions in many types of cells. In cultured hepatocytes, NO is produced by inducible NO synthase (iNOS) in response to interleukin (IL)-1beta. Cis-controlling elements and transcription factors which were involved in iNOS gene expression in hepatocytes have been unclear. RESULTS: We measured the transcriptional activity of the human iNOS gene promoter fused to the firefly luciferase gene in primary cultured rat hepatocytes. The luciferase assay of 5' deleted promoters revealed that the region from -365 to the transcription initiation site is required for the promoter activity of the iNOS gene. Mutations of a CCAAT/enhancer-binding protein (C/EBP)-binding site, namely the A-activator-binding site (AABS), and a nuclear factor (NF)-kappaB-binding site within this region, markedly decreased the promoter activity. Transfection of C/EBPbeta liver-enriched activator protein (LAP) or NF-kappaB (RelA + p50) activated the iNOS promoter, and transfection of LAP and NF-kappaB further activated it synergistically. In addition, either mutation of AABS and the NF-kappaB-binding site markedly reduced the basal promoter activity and the transactivation by LAP, NF-kappaB, and a combination of LAP and NF-kappaB. Electrophoretic mobility shift assays showed that C/EBPbeta was bound to AABS. CONCLUSION: These results demonstrate that C/EBPbeta may involve iNOS gene expression synergistically with NF-kappaB in primary cultured rat hepatocytes.

Prevention of ornithine cytotoxicity by proline in human retinal pigment epithelial cells.

PURPOSE: To investigate the relationship between ornithine-delta-aminotransferase (OAT) deficiency and ornithine accumulation and the specific degeneration of retinal pigment epithelial (RPE) cells in gyrate atrophy. METHODS: Human RPE cells, human hepatoma cells, and human fibroblast cells were treated with 5-fluoromethylornithine (5-FMOrn), a specific irreversible inhibitor of OAT. Ornithine cytotoxicity was determined by using a [3H]thymidine incorporation assay and immunohistochemical staining for cytokeratin. The effects of various metabolites of ornithine and arginine, such as creatine, creatine phosphate, I-delta 1-pyrroline-5-carboxylic acid (L-P5C), and proline, which may be deficient in gyrate atrophy on RPE cell damage by ornithine, were determined by the same procedures. RESULTS: When the human RPE cells, HepG2 hepatoma cells, and WI-38 fibroblast cells were treated with 0.5 mM 5-FMOrn for 30 minutes, which inactivated OAT, ornithine exhibited severe time- and dose-dependent inhibition of DNA synthesis in the human RPE cells but not in the HepG2 hepatoma cells or WI-38 fibroblast cells. The inhibition of DNA synthesis was accompanied by drastic changes in morphologic appearance, disorganization of the cytoskeleton, and cell death. Ornithine or 5-FMOrn alone did not exhibit such cytotoxicity to the RPE cells. Proline prevented the cytotoxicity of ornithine. CONCLUSIONS: These findings suggest that an elevated level of ornithine combined with an increased sensitivity to ornithine as a result of OAT deficiency may be crucial to the specific RPE degeneration in gyrate atrophy. They suggest also that abnormalities of proline metabolism may be involved in the progress of gyrate atrophy.

Expression of murine novel zinc finger proteins highly homologous to Drosophila ovo gene product in testis.

We have cloned two isoforms of cDNAs encoding novel zinc finger proteins. One form encodes a 274-amino acid protein containing an acidic amino acid and serine-rich domain and a zinc finger domain which shows high sequence homology to that of Drosophila Ovo protein. The other form encodes a 179-amino acid protein containing only the zinc finger domain. Expression of both proteins possessing an antigenic epitope in COS cells revealed that they are localized in the nucleus. The 1.3-kbp mRNAs are predominantly expressed in testis, and the expression increases from 3 weeks postnatal, implying that these proteins may play important roles in the development of the testes.

Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus.

We reported here purification and characterization of a novel heptadecapeptide in bovine brain as an endogenous ligand for ROR-C, an opioid receptor homologue cloned from rat cerebrum. The amino acid sequence of the peptide that we purified is identical to those recently identified as nociceptin in rat brain and orphanin FQ in porcine brain. The peptide inhibited the forskolin-induced cyclic AMP accumulation in ROR-C expressing Chinese hamster ovary cells. Studies on inhibitory activity of cyclic AMP accumulation and Northern blot analysis showed that the peptide and its precursor mRNA are present in a number of brain regions, less abundant in the spina cord, and negligible in the cerebellum. In situ hybridization analysis revealed that hybridization-positive neurons were distributed in the superficial layer (lamina I) of the dorsal horn and were also interspersed between the tract of Lissauer in the spinal cord. Intrathecal administration of the peptide into conscious mice induced allodynia, a pain response to innocuous tactile stimuli, in a beli-shaped manner. These results demonstrate that the peptide exists in the brain and spinal cord and plays an important role in pain transmission.

Hormonal regulation of prostaglandin F2 alpha receptor gene expression in mouse ovary.

In this study we examined regulation by pituitary gonadotropins of the prostaglandin F2 alpha (PGF2 alpha) receptor gene expression in the mouse ovary. Administration of pregnant mare serum gonadotropin (PMSG) to 35-day-old mice in the diestrus phase stimulated the ovary and enhanced the production of progesterone at 1 h PMSG also increased the ovarian PGF2 alpha receptor mRNA level in a time-dependent manner, reaching a sixfold maximum at 1 h. These actions of PMSG were mimicked by human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), and cholera toxin, all of which elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP). In situ hybridization revealed that PGF2 alpha receptor mRNA was localized to the corpus luteum, but the intensity of staining varied among corpora lutea in the same ovary. Exogenous PGF2 alpha inhibited the PMSG-stimulated progesterone production. These results demonstrate that gonadotropins may induce the expression of the PGF2 alpha receptor gene in luteal cells of the corpus luteum, probably by acting through a cAMP-mediated pathway, and that expression of the PGF2 alpha receptor may be functionally associated with the decrease in serum progesterone level.

Characterization of prostaglandin F2 alpha production in pregnant and cycling mice.

To clarify the regulation of prostaglandin F2 alpha (PGF2 alpha) production in vivo in mice during pregnancy and the estrous cycle, we injected [3H]PGF2 alpha i.v. into female mice and determined the structures of three urinary metabolites by gas chromatography-mass spectrometry. The compounds were 5,7,11-trihydroxy-tetranor-prosta-9-enoic acid (FUM-I), 5,7,11-trihydroxy-tetranor-prostanoic acid (FUM-II), and 5,7-dihydroxy-11-keto-tetranor-prostanoic acid (FUM-III). The major metabolite, FUM-III, increased four-fold at the term of pregnancy and transiently during diestrus of the estrous cycle. Consistent with the increase in FUM-III, immunoblot analysis with anti-bovine PGF synthase antiserum demonstrated that a 36-kDa protein band corresponding to PGF synthase increased in the uterus at late pregnancy and during diestrus. These results suggest that PGF2 alpha production may increase at term and change during the estrous cycle and is associated with an increase in uterine PGF synthase concentrations.

Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF.

BACKGROUND: The survival and differentiation of motoneurons during embryonic development, and the maintenance of their function in the postnatal phase, are regulated by a great variety of neurotrophic molecules which mediate their effects through different receptor systems. The multifactorial support of motoneurons represents a system of high security, because the inactivation of individual ligands has either no detectable, or relatively small, atrophic or degenerative effect on motoneurons. RESULTS: Leukaemia inhibitory factor (LIF) has been demonstrated to support motoneuron survival in vitro and in vivo under different experimental conditions. However, when LIF was inactivated by gene targeting, there were no apparent changes in the number and structure of motoneurons and no impairment of their function. The slowly appearing, relatively mild degenerating effects in motoneurons that resulted from ciliary neurotrophic factor (CNTF) gene targeting were substantially potentiated by simultaneous inactivation of the LIF gene, however. Thus, in mice deficient in LIF and CNTF, the degenerative changes in motoneurons were more extensive and appeared earlier. These changes were also functionally reflected by a marked reduction in grip strength. CONCLUSIONS: Degenerative disorders of the nervous system, in particular those of motoneurons, may be based on multifactorial inherited and/or acquired defects which individually do not result in degenerative disorders, but which become apparent when additional (cryptic) inherited disturbances or sub-threshold concentrations of noxious factors come into play. Accordingly, the inherited inactivation of the CNTF gene in a high proportion of the Japanese population may represent a predisposing factor for degenerative disorders of motoneurons.

Interleukin 1 beta markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells.

To investigate whether a single inflammatory cytokine could stimulate nitric oxide formation in the absence of other cytokines or lipopolysaccharide (LPS), NO was measured by the redox chemiluminescence method in primary cultured rat hepatocytes and in rat Kupffer cells. Interleukin (IL) 1 beta, but neither IL-6, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), nor LPS stimulated NO formation in a dose-dependent manner and induced half-maximal effects at 30 pmol/L. Maximal stimulation was achieved at 12 to 16 hours after the addition of 1I nmol/L of IL-1 beta, and was 50- to 60-fold above basal levels in rat hepatocytes. The combined effect of these cytokines with LPS or IFN-gamma on NO formation was also examined. Neither LPS nor IFN-gamma affected the IL-1 beta-induced NO formation. TNF-alpha, however, stimulated IL-1 beta-induced NO formation, while IL-6 inhibited it, although independently these cytokines had no effect on NO formation. None of the cytokines tested stimulated NO formation in cultured rat Kupffer cells. In hepatocytes, the NO formation induced by IL-l beta was blocked by both the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) and by IL-1 receptor antagonist (IL-1ra). Furthermore, IL-1 beta markedly increased NOS activity, and this increase in activity was accompanied by the expression of inducible NOS (iNOS) messenger RNA (mRNA). This study clearly demonstrated that IL-1 beta markedly stimulates NO formation in hepatocytes, in the absence of other cytokines or LPS.

Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation.

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.

Disruption of the CNTF gene results in motor neuron degeneration.

CNTF is a cytosolic molecule expressed postnatally in myelinating Schwann cells and in a subpopulation of astrocytes. Although CNTF administration prevents lesion-mediated and genetically determined motor neuron degeneration, its physiological function remained elusive. Here it is reported that abolition of CNTF gene expression by homologous recombination results in a progressive atrophy and loss of motor neurons in adult mice, which is functionally reflected by a small but significant reduction in muscle strength.

Enhancement of preprotachykinin A gene expression by adjuvant-induced inflammation in the rat spinal cord: possible involvement of substance P-containing spinal neurons in nociception.

Effects of adjuvant-induced inflammation on the biosynthesis of substance P in the rat nervous system were examined by measuring the levels of mRNA encoding preprotachykinin A (PPT-A, the precursor protein of substance P). Following injection of adjuvant into the bilateral hind paws, the levels of PPT-A mRNA were significantly increased in the dorsal root ganglia at L4-L6 levels and the lumbar spinal cord, but not in the striatum, midbrain and medulla oblongata. After the unilateral injection of adjuvant which produced inflammation only in the injected hind paw, increase in the mRNA level was observed only on the treated side of the spinal cord. These results suggest that biosynthesis of substance P in the spinal and primary sensory neurons was increased by adjuvant-induced inflammation with hyperalgesia. Substance P-containing spinal neurons may be involved in processes related to pain.

Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination.

D-Amino acid aminotransferase was found in several thermophilic Bacillus species and purified to homogeneity from the best producer, Bacillus sp. YM-1, which was newly isolated from a sauna dust. The enzyme has a molecular weight of about 62,000 and consists of two subunits identical in molecular weight (30,000). It catalyzes transamination between various D-amino acids and alpha-keto acids, although the substrate specificity is narrower than the enzyme from the mesophile, Bacillus sphaericus (Yonaha, K., Misono, H., Yamamoto, T., and Soda, K. (1975) J. Biol. Chem. 250, 6983-6989). The Bacillus sp. YM-1 enzyme is most active at 60 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 20 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 3 amino acids. The amino acid sequence in the vicinity of the lysyl residue, Lys(Pxy), that binds pyridoxal 5'-phosphate was determined as Cys-Asp-Ile-Lys(Pxy)-Ser-Leu-Asn-Leu-Leu-Gly-Ala-Val-Leu-Ala-Lys- from the pyridoxyl peptide obtained by digestion with trypsin. The active site sequence is markedly different from those of L-amino acid aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes.

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases.

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.