Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease.

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.

Arthroscopically-assisted reduction of intra-articular fractures and soft tissue management of distal radius.

Arthroscopy was used to help to reduce intra-articular fractures of the distal radius and treat soft tissue injuries in 33 acute cases. The fractures were treated by reduction under arthroscopic control and percutaneous fixation either with or without external fixation. The triangular fibrocartilage complex (TFCC) was torn in 18 of 33 patients (54%). All tears were peripheral and were repaired with arthroscopic procedures. Scapholunate (SL) ligament injuries prevailed in six (18%) patients; most of them exhibited instability in the SL joint. They received SL ligament debrided and transfixed with K-wires. Four (12%) of the patients suffered lunotriquetral (LT) ligament injuries; three of them also received transfixation with K-wires. Six (18%) of the patients exhibited chondral fractures. All fractures healed without measurable incongruity of joint surface and at follow-up (24 to 36 months), 11 patients displayed excellent results and 22 patients displayed good results according to the Mayo modified wrist score.

Regulation of insulin-like growth factor binding protein-5 mRNA abundance in rat intestinal smooth muscle.

IGF-I increases abundance of IGFBP-5 mRNA in rat intestinal smooth muscle cells (RISM), and IGFBP-5 protein in RISM conditioned media. The translational blocker, cycloheximide, decreased the abundance of IGFBP-5 mRNA to undetectable levels, suggesting that IGFBP-5 mRNA integrity is linked to protein synthesis. We studied the mechanism of IGF-I's effect on IGFBP-5 mRNA, and the role of cytoplasmic proteins in modulating IGFBP-5 mRNA abundance. Anisomycin, emetine, and puromycin abolished IGFBP-5 mRNA as seen with cycloheximide. Cycloheximide had a dose- and time-dependent effect on IGFBP-5 mRNA. IGF-I increased IGFBP-5 nuclear transcripts by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that IGF-I acts at least partially by increasing IGFBP-5 mRNA transcription. Protein synthesis inhibitors did not affect IGFBP-5 nuclear transcripts, therefore, they affect only mature mRNA. The IGFBP-5 mRNA 3' and 5' UTRs were cloned and their sequences searched for adenosine-uridine rich elements (AUREs), elements shown to regulate RNA stability. RNA mobility gel shift assay showed two protein activities that bind to nt 922 to 2076 of the 3' UTR, a region that contains an AURE. One protein activity (BA2) was decreased in cytoplasmic extracts from cycloheximide-treated RISM. These data demonstrate that IGFBP-5 mRNA integrity is dependent on protein synthesis. The 3' UTR of IGFBP-5 contains elements shown to bind proteins important for RNA stability regulation. This region binds RISM cytoplasmic proteins, and may mediate the dramatic effect of cycloheximide on IGFBP-5 abundance. RNA-protein interactions may be important to IGFBP-5 mRNA stability and ultimately, to IGFBP-5 actions.

Identification of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of rat Leydig cells as type II retinol dehydrogenase.

Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5alpha-reductase, 2) conversion of DHT to 5alpha-androstane-3alpha, 17beta-diol (3alpha-DIOL) by the reductive activity of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), and 3) conversion of 3alpha-DIOL by an oxidative 3alpha-HSD activity. While the type I 3alpha-HSD enzyme (3alpha-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3alpha-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3alpha-HSD1, and a microsomal NAD(H)-dependent activity. The two activities were separable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3alpha-HSD in rat Leydig cells, degenerate primers were used to amplify a number of short-chain alcohol dehydrogenases. Sequence analysis of cloned PCR products identified retinol dehydrogenase type II (RoDH2) as the prevalent species in purified Leydig cells. RoDH2 cDNA was subcloned into expression vectors and transiently transfected into CHOP and COS-1 cells. Its properties were compared with transiently transfected 3alpha-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3alpha-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3alpha-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3alpha-HSD cDNA produced a protein that was exclusively a 3alpha-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 3alpha-HSD oxidation and reduction reactions that were NAD(H) dependent, and the enzyme activities were located in the microsomes. Type I 3alpha-HSD also catalyzed both oxidation and reduction, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3alpha-HSD and RoDH2 have distinct 3alpha-HSD activities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells.

Dexamethasone regulation of the rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase gene.

Rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD), a member of the aldo-keto reductase superfamily, inactivates circulating steroid hormones and may contribute to the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) by oxidizing trans-dihydrodiols to reactive o-quinones with the concomitant generation of reactive oxygen species. The 3alpha-HSD/DD gene has been cloned, and its 5'-flanking region contains a negative response element (NRE; -797 to -498 bp) that may repress constitutive expression by binding to Oct transcription factors. Upstream from the NRE are three distal imperfect glucocorticoid response elements (GRE1, GRE2, and GRE3); in addition, a proximal imperfect GRE (GRE4) is adjacent to an Oct binding site in the NRE. When rat hepatocytes were cultured on Matrigel and exposed to dexamethasone (Dex), steady state levels of 3alpha-HSD/DD mRNA were increased 4-fold in a dose-dependent manner, yielding an EC50 value of 10 nM. Time to maximal response was 24 hr, and the effect was blocked with the anti-glucocorticoid RU486. Measurement of the half-life of 3alpha-HSD/DD mRNA, with and without Dex treatment, indicated that the increase in steady state mRNA levels was not due to increased mRNA stability. By contrast, nuclear run-off experiments using nuclei obtained from Dex-stimulated hepatocytes indicated that Dex increased transcription of the rat 3alpha-HSD/DD gene. Tandem repeats of the imperfect GRE1, GRE2, GRE3, and GRE4 were inserted into thymidine kinase-chloramphenicol acetyl-transferase vectors and cotransfected with the human glucocorticoid receptor into human hepatoma cells. On treatment with Dex, maximal trans-activation of the chloramphenicol acetyl-transferase reporter gene activity was mediated via the proximal GRE (GRE4). These data imply that GRE4 is a functional cis-element and that binding of the occupied glucocorticoid receptor to this element increases 3alpha-HSD/DD gene transcription. A model is proposed for the positive and negative regulation of the rat 3alpha-HSD/DD gene by the glucocorticoid receptor and Oct transcription factors, respectively.

IGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth muscle.

Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in intestines of rats with peptidoglycan-polysaccharide enterocolitis by Northern analysis and in situ hybridization. IGFBP-5 mRNA was increased 2.4 +/- 0.5-fold in inflamed rat colon compared with controls and was highly expressed in smooth muscle. Cultured rat intestinal smooth muscle cells were used to study the regulation of IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased IGFBP-5 mRNA (1.9 +/- 0.1-fold) and collagen type alpha1(I) mRNA (1.6 +/- 0.2-fold) in cultured smooth muscle cells. IGF-I induced a dose- and time-dependent increase in IGFBP-5 in conditioned medium by Western ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5 mRNA decay rate after transcriptional blockade. Cycloheximide abolished IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by intestinal smooth muscle and is increased during chronic inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in intestinal smooth muscle cells.

AP-4- and AP-5-like proteins from mouse L cells are trans-activators and bind to the GT-II region of SV40 early TRE in a mutually exclusive manner.

Plasmids containing the cat reporter gene, transcription of which is directed by deletion mutants of the SV40 early region transcriptional regulatory element (SV40E TRE), were transfected into mouse L cells to determine the DNA motifs of SV40E TRE responsible for maximal gene expression. One deletion mutant, pSVE305, demonstrated a 50% reduction in CAT activity as compared to pSVE338, suggesting the importance of these 33 bp in directing efficient gene expression in mouse L cells. Introduction of triplet point mutations in this region and subsequent transfection studies in mouse L cells revealed three sites which were responsible for the reduction of CAT activity. These three mutations were located in the middle of the binding sites of three trans-activators: AP-3, AP-4 and AP-5. While the levels of CAT activity directed by SV40E TRE deletion mutants were similar in both HeLa and mouse L cells, the profiles of point mutants were different, suggesting that the activating ability of each nuclear factor is different from that of its counterpart in these two cell lines. Electrophoretic mobility shift assays (EMSA) demonstrated that binding of AP-4- and AP-5-like proteins of mouse L and HeLa cells to the GT-II motif occurs in a mutually exclusive manner. Furthermore, we observed a 'reverse competition' binding phenomenon which suggested a unique relationship between AP-4- and AP-5-like proteins of mouse L cells to the GT-II motif. Proteolytic mobility-shift analyses showed that an AP-5-like protein was more resistant to proteolytic digestion than an AP-4-like protein of mouse L cells.

Rat dihydrodiol dehydrogenase: complexity of gene structure and tissue-specific and sexually dimorphic gene expression.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes a novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism in which trans-dihydrodiols (proximate carcinogens) are oxidized to reactive o-quinones which are cytotoxic and genotoxic. In this study, the complementary DNA for rat liver DD was used to examine the structure and regulation of the DD gene. Southern analysis of rat genomic DNA confirmed that DD is a member of the multigene aldo-keto reductase superfamily. Conservative estimates indicate that the rat DD gene is at least 20-25 kilobases in length. Northern analysis showed that the rat liver transcript was 2.4 kilobases whereas the complementary DNA contains an open-reading frame of 966 nucleotides. Primer extension of male and female polyadenylated RNA indicated that the major transcription start sites are only 53 and 54 base pairs upstream from the translation start site, confirming that the RNA has a very long 3'-untranslated region. In male and female tissues, 2.4 kilobase transcripts predominate in liver, small intestine, and lung, which is consistent with a role for the enzyme in PAH metabolism. Transcripts were also detected in male (prostate)- and female (ovary, mammary gland, and uterus)-specific tissues. In the ovary, two transcripts were observed of 2.4 and 1.4 kilobases in length. Using benzenedihydrodiol as a model substrate for PAH trans-dihydrodiols, highest levels of DD activity were observed in the liver and small intestine of both sexes. Enzyme activity is 2.5-fold higher in the female liver versus the male liver. This sexual dimorphism can be explained by increases in the DD mRNA and enzyme protein as measured by dot-blot and immunotitration analyses, respectively. The latter measurements indicate that DD represents 1.0% of the soluble protein in female liver but is only 0.5% of the soluble protein in male liver. Hormonal ablation (ovariectomy and hypophysectomy) abolishes the sexual dimorphism observed in levels of DD mRNA, enzyme protein, and enzyme activity. Administration of estrogens to males is sufficient to establish the female pattern of gene expression. These data indicate that DD gene expression is hormonally regulated, that estrogens exert their effect at the level of the mRNA, and that aldo-keto reductases involved in PAH metabolism may have their expression regulated by female sex hormones.

The SV40 early transcriptional regulatory element is unable to direct gene expression in pituitary GH-3 cells.

The SV40 early (SV40E) transcriptional regulatory element (TRE) is able to direct heterologous gene expression in a variety of eukaryotic cell lines. This ability is conferred, in part, by the presence of several cis-elements. Transfection studies, mutational analyses, and in vitro DNA binding assays have demonstrated that the SV40E TRE is capable of interacting with several cellular transcription (trans) factors. In the present study, we have investigated the inability of the SV40E TRE to direct gene expression in cultured rat anterior pituitary GH-3 cells. Gel shift analysis demonstrated that nuclear factors within these cells can recognize and specifically bind to DNA containing SV40 enhancer sequences. Surprisingly, we have found that both HeLa and GH-3 cells possess relatively equal quantities of Sp1-specific RNA; however, a dramatic decrease in Sp1 protein was seen in GH-3 cells. Transfection studies utilizing CAT reporter plasmids revealed that the intact SV40E TRE is inactive in these cells, and that subsequent deletion of a region(s) where nuclear factor binding occurs does not result in detectable levels of gene expression. Thus, removal of cis-sites potentially involved in repressor binding does not result in activation of the SV40E TRE in these cells. Subcloning an SV40 enhancer fragment upstream of a heterologous TK promoter yielded chimeric TREs that could direct high levels of gene expression in HeLa but not GH-3 cells. Therefore, the prototypic SV40 enhancer, in the context of GH-3 cells, cannot enhance gene expression.

Importance of SV40 early 5' distal sequences in directing heterologous gene expression.

The chloramphenicol acetyltransferase-encoding reporter gene (cat) is used extensively in assessing the ability of transcriptional regulatory elements (TRE) to direct gene expression in eukaryotic cells. Two commonly utilized plasmids contain the cat coding sequences under the transcriptional control of the Rous sarcoma virus LTR (pRSVcat) or simian virus 40 early (SV40E) promoter (pSV2cat). In the present study, we have recloned the RSV-LTR and SV40E TRE into a pUC18 vector. Direct comparison of these TRE in different plasmid vectors, as well as reevaluation of their relative level of cat expression revealed: (1) a small but significant increases in SV40E-directed reporter gene expression was observed when the TRE was inserted into the pUC18 vector; and (2) a significant increase in SV40E-directed gene expression was realized by inclusion of the 69-bp 5' of the sequences present in pSV2cat. These distal sequences are required for maximal activity of the SV40 TRE in the cell lines tested.

Efficient expression of unfused human alpha D-interferon in Escherichia coli using overlapping termination and initiation codons (TGATG) in its signal sequence.

A plasmid carrying the lambda PL promoter was constructed to express efficiently unfused human alpha D-interferon (HuIFN-alpha D) in Escherichia coli using a TGATG site in its signal sequence, which occurs also in the lambda DNA sequence. The unfused nature of HuIFN-alpha D expressed by pBV867 in E. coli (BMH 71-18) was confirmed by the following evidence: first, the purified IFN showed a single band of 19.5K in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); a 27K band, representing lambda N-HuIFN-alpha D fusion protein, was not detected. Second, the peak of IFN activity coincided with the 19.5K protein. Third, the peak of absorbent material for human leukocyte IFN antibody coincided with that of IFN activity. Finally, amino-terminal sequencing of purified IFN demonstrated an unfused HuIFN-alpha D. This suggests that E. coli is able to process the signal sequence of HuIFN-alpha D. Studies on the mechanism of "translational coupling" initiation of gene expression were carried out by the construction of two hybrid plasmids and titration of the IFN activities produced by them. The level of expression by the ATG-TGATG initiation mode was found to be six times higher than that of the single ATG mode.

Variation in the HindIII restriction fragments of DNA from the Chinese Tian Tan strain of vaccinia virus.

HindIII fragments D to P of DNA from a Chinese vaccine strain (Tian Tan) of vaccinia virus have been molecularly cloned into the plasmid pAT153 at the unique HindIII site. The Chinese strain DNA differs from a non-vaccine American strain (WR) in having an additional HindIII fragment (P). Twelve HindIII D clones and 12 HindIII F clones of the Chinese strain were analysed by digestion by EcoRI, BamHI, PstI and XhoI. Two forms of D (designated a and b) and of F (a and b) were demonstrated. In each the differences were detected as the presence of an additional EcoRI site, in Da and Fa. The HindIII Fa and Fb fragments of the Chinese strain were shown to differ significantly from the WR strain in their restriction site maps.

Enhancement of spontaneous VCA and EA induction in B95-8 cells and EA induction in Raji cells treated with human leukocyte interferon.

The purpose of this study was to determine the antiviral effect of human leukocyte interferon on Epstein-Barr virus (EBV) viral capsid antigen (VCA) and early antigen (EA) induction in B95-8 and Raji cells. Interferon made at the Institute of Virology, Beijing, and that provided by Dr. Cantell gave unexpected results. Both interferon preparations markedly enhanced spontaneous VCA and EA induction in B95-8 cells and EA induction in Raji cells simultaneously treated with croton oil and n-butyrate. Interferon treatment alone had no effect on EA induction in Raji cells. Thus, the effect of interferon on EA and VCA induction was related to the type of EBV infection, i.e., productive or latent. The enhancing activity of interferon could only be partially inhibited by retinoid 7901. It is suggested that the mechanism for enhancement of EA induction by interferon is different from that of EA induction in Raji cells by croton oil and n-butyrate.