Hypoxia extends the life span of vascular smooth muscle cells through telomerase activation.

Chronic hypoxia induces smooth muscle cell proliferation and vessel wall remodeling in the vasculature of the lung. One well-characterized component of the hypoxic response is transcriptional activation of genes encoding vascular smooth muscle cell (VSMC) mitogens. We report here that chronic hypoxia can also prolong the growth of human VSMC by inducing telomerase activity and telomere stabilization. We demonstrate that hypoxia induced phosphorylation of the telomerase catalytic component (TERT) and sustained high levels of TERT protein expression in VSMC compared to normoxia. Furthermore, inhibition of telomerase shortened cell life span in hypoxic cultures, whereas constitutive expression of TERT extended the life span of cells under normoxic conditions. Our data indicate that hypoxic induction of telomerase activity could be involved in long-term growth of VSMC and may thus contribute to human vascular disorders.

An upstream regulatory domain of avian tumor virus long terminal repeat is required for the expression of a procaryotic neomycin gene in eucaryotic cells.

The regulatory elements present in the long terminal repeat (LTR) of avian sarcoma virus DNA were analyzed by recombinant DNA techniques coupled with DNA-mediated gene transfer in avian as well as mammalian cells. For this purpose, the neomycin resistance gene from transposon Tn5 was inserted downstream from the avian sarcoma virus LTR, and the recombinant plasmid DNA was introduced into cells by the calcium phosphate technique. Cells resistant to the drug G-418 were selected. Analysis of the RNA transcripts made in vivo in these transformants indicated that initiation and termination of the transcripts occurred in the LTR sequences. Deletions were then introduced into the LTR, and their effect on transcription was also studied. These results allowed us to identify a strong regulatory sequence between nucleotides -299 and -114 in the LTR of avian sarcoma virus.

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

Binding of Escherichia coli RNA polymerase to a specific site located near the 3'-end of the avaian sarcoma virus genome.

We have isolated a unique 35 base pair region of avian tumor virus DNA that binds specifically to Escherichia coli RNA polymerase holoenzyme. Studies with various size classes of viral DNA coupled with restriction enzyme mapping data indicate that the binding site is located in the large terminal repeat of the viral genome and is within the first 50 nucleotides of the heteropolymeric region corresponding to the 3'-end of the virion RNA.

Hypoxia induces macrophage inflammatory protein-2 (MIP-2) gene expression in murine macrophages via NF-kappaB: the prominent role of p42/ p44 and PI3 kinase pathways.

We have previously reported that hypoxia induces a pronounced inflammatory response in the mouse lung associated with elevated levels of specific chemokines. To further explore the mechanisms involved in lung inflammation, we exposed RAW 264.7 cells as well as mouse primary macrophages to hypoxia and analyzed chemokine gene expression. Among the genes examined, macrophage inflammatory protein-2 (MIP-2) expression was prominently induced by hypoxia both at the mRNA and the protein level. When RAW 264.7 cells were transfected with a panel of plasmids harboring a luciferase marker gene under the control of wild-type or mutant variants of the MIP-2 gene promoter, a strong hypoxic induction of expression (9- to 17-fold) was observed. This induction was abolished by a mutation targeted to an NF-kappaB binding site in the MIP-2 promoter. Concordantly, specific NF-kappaB binding to the cognate sequence was enriched in nuclear extracts from hypoxic but not normoxic RAW 264.7 cells. The mechanism of MIP-2 gene induction by hypoxia was further characterized using inhibitors of signaling kinases. Inhibition of the p42/p44 and PI3 kinases but not p38 MAPK abolished the NF-kappaB-driven upregulation of MIP-2 gene expression by hypoxia. This attenuation of the NF-kappaB response to hypoxia did not involve decreased nuclear NF-kappaB abundance but correlated with diminished transactivation potential of the p65 subunit. Our results indicate that the hypoxic signal for induction of MIP-2 gene expression is implemented through enhanced NF-kappaB activity and transmitted along the p42/44 and PI3 kinase pathways.

Cloning of avian tumor virus DNA fragments in plasmid pBR322: evidence for efficient transcription in E. coli from a virus-coded promoter.

Two avian tumor virus DNA fragments of 4.2 and 3.2 kb were inserted into pBR322 in the two possible orientations for each fragment. In the Escherichia coli host cells, RNA polymerase initiates transcription of large quantities (up to 0.5 to 1% of total E. coli RNA) of virus-specific RNA in the recombinant plasmids carrying the 4.2-kb fragment (pATV-6) but not in pATV-2 which contains the 3.2-kb fragment. Two SacI cleavage sites flank the putative promoter in the 4.2-kb viral insert. Deletion in the 1.2-kb SacI fragment obliterated the ability of pATV-6 to synthesize viral RNA. Digestion of the 1.2-kb SacI fragment with PvuI generates two fragments of 0.63 and 0.57 kb. Deletion of the 0.57-kb but not the 0.63-kb PvuI-SacI fragment completely eliminated the ability of the recombinant to synthesize viral RNA. These results strongly suggest that viral RNA in E. coli transcription is indeed initiated at a size present in the viral genome and that this site is localized in the 0.57-kb PvuI-SacI fragment.

An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli.

A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.

Effect of heme oxygenase-1 overexpression in two models of lung inflammation.

An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and bilirubin, downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment or by repressing the induction of cytokines and chemokines. In the present study we used genetically engineered mice that express high levels of a human cDNA HO-1 transgene in lung epithelium to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) challenge. We found that both mRNA and protein levels of specific cytokines and chemokines were significantly elevated in response to hypoxia in the lungs of wild-type mice after 2 and 5 days of exposure but significantly suppressed in the hypoxic lungs of transgenic mice, suggesting that inhibition of these cytokines was caused by overexpression of HO-1. However, LPS treatment resulted in a very pronounced increase in mRNA levels of several cytokines in both wild-type and transgenic mice. Despite the high mRNA levels, significantly lower cytokine protein levels were detected in the bronchoalveolar lavage of HO-1 overexpressing mice compared with wild type, indicating that HO-1 leads to repression of cytokines in the airway. These results demonstrate that HO-1 activity operates through distinct molecular mechanisms to confer cytoprotection in the hypoxic and the LPS models of inflammation.

Regulatory elements controlling chorion gene expression are conserved between flies and moths.

Flies and moths are approximately as distant phylogenetically as are mammals and birds. In terms of morphology, physiology and biochemistry, the complex proteinaceous eggshell or chorion differs substantially in these two insect groups, which are typified by Drosophila melanogaster and Bombyx mori. The major chorion proteins of moths are encoded by two families of genes, A and B, which have no obvious homologues in flies. Unlike Drosophila, where chorion genes are oriented in tandem, moths show mostly chorion gene pairs (A plus B) that are divergently transcribed and coordinately expressed. The 5' ends of the paired genes are separated by a DNA segment of only 300 +/- 50 base pairs, which may well include at least some of the cis-regulatory elements necessary for gene expression. Despite these differences, we have tested whether moth chorion genes might be expressed in flies. Cloned DNA fragments bearing moth chorion genes were introduced into the Drosophila germ line by P-element-mediated transformation. Analysis of RNAs from transformed lines revealed that the genes are expressed with correct sex, tissue and temporal specificity, resulting in the accumulation of abundant moth chorion transcripts in late fly follicles.

Transgenic regulation of moth chorion gene promoters in Drosophila: tissue, temporal, and quantitative control of four bidirectional promoters.

Bidirectional chorion gene promoter regions from three silkmoth species, Bombyx mori, Antheraea pernyi, or Antheraea polyphemus (members of two different moth families), were tested for their ability to transcriptionally activate a bacterial marker gene (chloramphenicol acetyltransferase) in transformant Drosophila. Relatively short 5' flanking DNA fragments (272-367 bp) of chorion gene pairs are sufficient to confer a high degree of tissue and choriogenic stage specificity of expression to the marker gene. Thus, significant conservation of molecular interactions controlling transcription during choriogenesis is observed between the distantly related orders, Lepidoptera and Diptera. However, quantitative and fine temporal regulation in the Drosophila host does not fully parallel the in situ regulation in moths, indicating that some regulatory protein-DNA interactions have diversified in the approximately 250 million years since the last common ancestor of these insect groups. Limited in vitro mutagenesis of a B. mori promoter DNA has shown that a central 189-bp region includes elements sufficient for the qualitative specificity of chorion-specific expression. The same experiments have shown that a previously identified essential element, centered on the TCACGT hexamer, is not sufficient for chorion-specific expression: an additional essential element or elements are found farther upstream, within a 112-bp DNA region. Comparisons of silkmoth and Drosophila chorion gene promoter sequences have identified some candidates for cis-acting elements involved in the developmental regulation of chorion gene expression.

Studies on the methylation of avian sarcoma proviruses in permissive and non-permissive cells.

The extent of methylation of several avian oncogenic proviruses was determined by using the restriction endonucleases HpaII and MspI. The results indicated that the transformation-defective proviruses (RAV-O or B77-td), which are exogenously introduced into avian host cells, were not methylated. However, endogenous proviruses (RAV-O) or ASV proviruses present in non-permissive host cells were found to be partly or completely methylated. The methyl-sensitive restriction endonuclease PvuI, which recognizes a unique site within the long terminal repeat in the ASV genome, failed to cleave proviruses present in several non-permissive host cells. From these results we suggest that modification of the sequence around the PvuI site results in reduced levels of transcription.

Carbon monoxide controls the proliferation of hypoxic vascular smooth muscle cells.

Excess vascular smooth muscle cell (VSMC) proliferation and contractility are key events in the pathophysiology of vascular disorders induced by hypoxia. We have recently reported that carbon monoxide (CO), produced by VSMC under conditions of hypoxia, can be a modulator of cGMP levels in both endothelial and smooth muscle cells. In this respect, some of the physiologic effects of CO in the vasculature parallel those of nitric oxide (NO), a well characterized regulator of vascular tone. We report here that under hypoxia, VSMC-derived CO is an important regulator of VSMC proliferation. Inhibiting CO formation or scavenging CO with hemoglobin increased VSMC proliferation in response to serum or to mitogens such as endothelin, whereas increasing CO production or exposing cells to exogenous CO lead to a markedly attenuated growth response. The effects of CO on VSMC proliferation correlated with changes in E2F-1 expression, the prototype member of a family of transcription factors that participate in the control of cell cycle progression. CO significantly suppressed E2F-1 expression, whereas, removal of CO from the cultures with hemoglobin lead to increased E2F-1 gene transcription, mRNA, and protein production as well as mRNA levels of c-myc, a target gene of E2F-1. Moreover, the actions of CO were mediated by the second messenger molecule, cGMP. Limiting VSMC growth by increasing the release of CO may represent a key event in the body's compensatory responses to hypoxia.

Modification of avian sarcoma proviral DNA sequences in nonpermissive XC cells but not in permissive chicken cells.

For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive chicken cells. Some of the endogenous viral sequences in the permissive cells were also methylated. No 5-methylcytosine could be detected in the unintegrated viral DNA.

Regulation of renal rK1-kallikrein in spontaneously hypertensive rats.

Urinary and renal rK1-kallikrein was studied in spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). It was demonstrated that the antiserum used for kallikrein radioimmunoassay (RIA) reacts with rK1- but not with rK7-protein. The specificity of the kininogenase assay was tested: rK7 had only 8% of the activity of rK1. Urinary kallikrein excretion by RIA was reduced by about two thirds in 5-week-old SHR compared WKY (11.5 versus 37.1 micrograms/24 h). On the contrary, the kidney content of rK1-kallikrein by RIA was increased by 40% in these rats (11.6 versus 8.4 ng/mg protein). The increase in kidney rK1 was confirmed by kininogenase assays. The same pattern of reduced urinary and increased renal rK1-kallikrein was observed in 8-week-old SHR rats. Kidney rK1-kallikrein mRNA tended to be lower (0.10 > p > 0.05) in SHR compared to WKY rats, suggesting that the increased kidney rK1 content is not due to increased rK1 synthesis. We hypothesize that the combination of high kidney content and low urinary excretion may be due to a defective mechanism for secretion of rK1 into the urine by tubular epithelial cells.

Studies on the structure and organization of avian sarcoma proviruses in the rat XC cell line.

The structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC cell line were studied by restriction endonucleases. The following observations were made: (i) the majority of the proviruses integrated randomly with respect to cell DNA; (ii) no gross deletions or rearrangements in the proviruses were observed; (iii) two types of proviruses (type I and type II) could be distinguished on the basis of restriction endonuclease cleavage sites; (iv) the virus rescued from these cells was derived from type II provirus, which has a novel EcoRI site between the env and pol genes; (v) most of the provirus units contained the src gene.

A short 5'-flanking DNA region is sufficient for developmentally correct expression of moth chorion genes in Drosophila.

Fusions with the bacterial gene for chloramphenicol acetyltransferase followed by P-element-mediated germ-line transformation in Drosophila have permitted localization of the DNA sequence that confers a high degree of developmental specificity on a pair of silkmoth eggshell (chorion) genes. The short, 272-base-pair, 5'-flanking region shared by the divergently transcribed genes is sufficient for developmentally appropriate expression when placed upstream of the chloramphenicol acetyltransferase gene, in either orientation. A highly conserved motif within that region, TCACGT, is essential for chorion-specific expression.

Ultraspiracle, a Drosophila retinoic X receptor alpha homologue, can mobilize the human thyroid hormone receptor to transactivate a human promoter.

We have analyzed the functional domains of the Drosophila orphan receptor Ultraspiracle (usp), a homologue of the vertebrate retinoic X receptor alpha, as well as the ability of heterodimers between usp and the thyroid hormone receptor beta (T3Rbeta) to transactivate the human apolipoprotein A-II (apoA-II) promoter. DNA binding assays demonstrated that heterodimers of usp and the human T3Rbeta can bind to the hormone response element (HRE) of the regulatory element AIIJ (-734 to -716) of the human apoA-II promoter. Cotransfection experiments have shown that the combination of usp and T3Rbeta can transactivate the human apoA-II promoter in COS-1 cells 7-8-fold in the presence of thyroid hormone (T3). The observed transactivation was not affected by the deletion of the amino-terminal residues 1-85 of usp, which represent a putative transactivation domain, suggesting that the function of usp is to recruit T3Rbeta. Furthermore, a mutant usp, with impaired DNA binding properties, can form heterodimers with T3Rbeta in vitro but has reduced ability to transactivate the human apoA-II promoter. A minimal thymidine kinase (tk) promoter driven by four AIIJ regulatory elements is repressed to 20% of its original activity by T3Rbeta and the repression is relieved by usp/T3Rbeta heterodimers. Deletion analysis demonstrated that factors bound to the regulatory elements AIIJ, AIIAB, and AIIH participate in the usp/T3Rbeta-mediated transactivation of the human apoA-II promoter. Similarly to element AIIJ, element AIIAB binds usp/T3Rbeta heterodimers, whereas element AIIH binds a COS-1 nuclear activity that is supershifted with anti-hepatic nuclear factor 1 antibodies. The findings suggest that optimal transactivation of the apoA-II promoter by usp/T3Rbeta heterodimers requires complex interactions between these heterodimers and factors bound to other regulatory elements. The observed transcriptional activation through heterodimer formation between nuclear receptors from species as divergent in the evolutionary scale as insects and mammals indicates that the functional domains of these proteins have been highly conserved.

Retinoic acid alters the expression of pattern-related genes in the developing rat lung.

Exogenous retinoids alter pattern formation and differentiation in many developing systems, such as limb, vertebrae, and central nervous system. Many of these effects are mediated by changes in expression of patterning genes such as Hox genes and Sonic hedgehog. We have previously shown that exogenous retinoic acid, administered to the embryonic rat lung in culture alters the structural pattern of the developing lung, suppressing formation of distal lung and favoring growth of proximal tubules. To determine whether these retinoic acid-induced changes in lung development were linked to alterations in pattern-related genes, we characterized the expression of Hoxa-2, Hoxb-6, and Sonic hedgehog mRNAs in vivo and in vitro, with or without 10(-5)M retinoic acid, by in situ hybridization and quantitative polymerase chain reaction. Each of these genes demonstrated unique timing and distribution of expression that was similar in vivo and in control cultured embryonic lungs. Hoxb-6 and Sonic hedgehog mRNAs both decreased during lung development in vivo or in vitro. From the patterns of mRNA expression we propose that Hoxb-6 is involved in distal airway branching while Hoxa-2 is involved in differentiation of proximal mesenchymal derivatives and vasculogenesis in the lung. RA upregulated all three genes, changing their developmental pattern of distribution and preventing the developmental decrease in Sonic hedgehog expression. We propose that RA acts to maintain high levels of expression of these and likely other pattern-related genes in a fashion that is characteristic of the immature lung, promoting continued formation of proximal lung structures and preventing formation of typical distal lung structures of the mature lung.

Retinoic acid induces changes in the pattern of airway branching and alters epithelial cell differentiation in the developing lung in vitro.

Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern formation in visceral organs. We investigated the effects of exogenous retinoic acid on the in vitro pattern of airway branching and on lung epithelial cell differentiation. Histology, [3H]thymidine autoradiographies and reverse transcriptase/polymerase chain reaction (RT/PCR) amplification were used to assess the effects of retinoids and the expression of lung epithelial markers of differentiation. We found that retinoic acid interferes, in a dose-dependent fashion, with the expression of epithelial genes that are found in distal segments of the fetal lung (surfactant-associated proteins SP-A, SP-B, and SP-C). At high concentrations, retinoic acid (RA) dramatically altered the developmental pattern of the lung, favoring growth of structures that resemble proximal airways and concomitantly suppressing distal epithelial buds. We hypothesize that this in vitro "proximalizing" effect on the developing lung may be related to alterations in the expression of pattern-related genes.