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R H Goldstein

Publications and source records attributed to R H Goldstein.

At least 37 records · Page 2Linked to original sources
PubMed1997

Colchicine does not ameliorate bleomycin-induced pulmonary injury in hamsters.

We have evaluated the effect of colchicine, a potential antifibrotic drug, on bleomycin-induced pulmonary inflammation in hamsters. Pulmonary injury was induced by a single intratracheal (IT) instillation of bleomycin (bleo). Four groups of male Syrian hamsters each received one of four treatments: (1) IT bleo and twice daily intraperitoneal (IP) injections of colchicine (col) starting one day before IT instillation of bleo (bleo-col); (2) IT bleo and IP injections of saline (bleo-sal); (3) IT saline and IP colchicine (sal-col); and (4) IT saline and IP saline (sal-sal). Animals were sacrificed 28 days after IT treatment. Lung injury was evaluated histologically, biochemically and by analysis of bronchoalveolar lavage (BAL) fluid. Treatment of hamsters with colchicine did not ameliorate the bleo-induced lung injury, as determined by a semiquantitative morphological index that assesses the severity and extent of the lung injury on a scale of 0-3. Lung hydroxyproline measurements and BAL fluid cell count were also similar in bleo-col compared to bleo-sal hamsters. Colchicine did not prevent the bleo-induced restriction expressed by volume displacement. These results indicate that colchicine does not ameliorate the bleo-induced lung inflammation and fibrosis and call for further controlled investigations to justify the use of this drug in pulmonary disorders.

Animals↗
PubMed1997

Bleomycin-induced lung injury is enhanced by interferon-alpha.

We have evaluated the effect of intraperitoneal (I.P.) injection of human recombinant interferon-2alpha (IFN-alpha) on Bleomycin-induced pulmonary injury in hamsters. Pulmonary injury was induced by a single intratracheal (I.T.) instillation of Bleomycin (Bleo). Six groups of male Syrian hamsters were treated as follows: 1) I.T. Bleo and daily I.P. injections of low-dose interferon-alpha (2 x 10(4) U), 2) I.T. Bleo and daily I.P. injections of high-dose interferon-alpha (10(5) U), 3) I.T. Bleo and I.P. injections of saline, 4) I.T. saline and I.P. low-dose IFN-alpha, 5) I.T. saline and I.P. high-dose IFN-alpha, 6) I.T. saline and I.P. saline. Animals were sacrificed 28 days after I.T. treatment. Lung injury was evaluated histologically and biochemically. Treatment of hamsters with low-dose but not high-dose IFN-alpha significantly augmented the Bleo-induced lung injury, as determined by a semiquantitative morphological index. Lung hydroxyproline measurements were highest in Bleo-low-dose-IFN-alpha followed by Bleo-high-dose-IFN-alpha and Bleo-Sal as compared to Sal-Sal and Sal-IFN-alpha controls. These results suggest that IFN-alpha augments Bleo-induced lung injury but that this effect is complex and does not follow a simple-dose-response pattern.

Animals↗
PubMed1996

Cell-specific expression of the alpha 1 (I) collagen promoter-CAT transgene in skin and lung: a response to TGF-beta subcutaneous injection and bleomycin endotracheal instillation.

Transgenic mice containing a rat collagen alpha 1 (I) promoter (3.6 kilobases) fused to the reporter gene chloramphenicol acetyl transferase (CAT) express the reporter gene parallel to endogenous gene in most connective tissues other than vascular tissue [Pavlin et al. (1992): J Cell Biol 116:227-236; Bedalov et al. (1994): J Biol Chem 269:4903-4909]. We have challenged transgenic mice with subcutaneous injections of transforming growth factor-beta (TGF-beta) or intratracheal instillation of bleomycin. In situ hybridization studies of skin revealed increased CAT expression in the papillary dermis of TGF-beta treated animals. In contrast, alpha 1 (I) collagen mRNA was expressed throughout the dermis including granulation tissue and reticular dermis. Therefore, the transgenic promoter responds to TGF-beta in a subset of dermal fibroblasts. Endotracheal instillation of bleomycin induces lung fibrosis which is thought to be mediated in part by TGF-beta. CAT gene expression in lungs was increased 6-8-fold at 2 weeks post bleomycin treatment. In situ hybridization studies revealed focal areas of cells expressing both CAT and collagen genes in the interstitium. However, most regions, especially around airways, contained a subset of cells expressing the endogenous gene with little or no CAT expression as judged by in situ hybridization. These cells could be myofibroblasts that require additional cis-acting elements to activate alpha 1 (I) collagen gene expression similar to smooth muscle cells.

Animals↗
PubMed1996

Regulation of lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions among TGF-beta, IL-1 beta, and prostaglandin E.

Prostaglandin E2, transforming growth factor-beta, and interleukin-1 beta variably regulate the expression of cyclooxygenase 1, cyclooxygenase 2, and lysyl oxidase in IMR90, human embryo lung fibroblasts. Prostaglandin E2 at 100 nM upregulates cyclooxygenase 1 mRNA by approximately three-fold while it downregulates lysyl oxidase mRNA levels. Notably, prostaglandin E2 suppresses the enhancing effect of TGF-beta on basal levels of lysyl oxidase mRNA. These changes in steady state mRNA levels reflect transcriptional level control, at least in part. Corresponding changes are seen in the protein levels of lysyl oxidase, cyclooxygenase 1 and cyclooxygenase 2 and catalytic activities of these enzymes, including net prostaglandin E2 synthesis. Cyclooxygenase 2 mRNA(t1/2, 30 min) is considerably less stable than that of cyclooxygenase 1 (t1/2, 4 h) while lysyl oxidase mRNA is unusually stable (t1/2 > 14 h). Taken together with the differing kinetics with which these genes respond to perturbation by these cytokines, the present results suggest a coordinated, autocrine-like mechanism of regulation of cyclooxygenase 1 and cyclooxygenase 2 and further point to the potential of their metabolic product, prostaglandin E2, to suppress the expression of lysyl oxidase in the inflammatory response to injury.

Cyclooxygenase 1↗
PubMed1996

Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-beta receptor: regulation by TGF-beta.

The type I transforming growth factor-beta (TGF-beta) receptors are serine/threonine kinases that are essential for the action of TGF-beta. In this paper, we describe the molecular cloning and expression of the R4/ALK5 human type I TGF-beta receptor promoter. DNA sequence analysis indicates that the promoter lacks a TATA and CAAT box but is highly GC-rich and contains putative Sp1 binding sites. The transcriptional start site is approx. 232 base pairs upstream of the AUG start codon. In human lung fibroblasts, TGF-beta induced a 3-fold increase in steady-state level for type I receptor mRNA. Exposure of cells transfected with a 618 bp promoter fragment to TGF-beta 1 up-regulated transcriptional activity indicating that a TGF-beta response element is contained within this region.

Activin Receptors, Type I↗
PubMed1996

Effect of okadaic acid on elastin gene expression in interstitial lung fibroblasts.

Okadaic acid (OA), a specific serine/threonine protein phosphatase inhibitor, downregulated tropoelastin formation and elastin mRNA levels in a dose-related and cycloheximide-sensitive fashion in cultured lung fibroblasts. Treatment with a tyrosine phosphatase inhibitor at high concentrations did not alter elastin mRNA levels, however. Nuclear run-on analysis indicated that OA primarily suppressed elastin gene expression through a transcriptional mechanism. In contrast to its effects on elastin expression, OA downregulated alpha 1(I) mRNA to significantly lesser degrees. The mechanism by which OA decreased elastin mRNA levels did not appear to involve protein kinase C or share the signaling pathway of IL-1 beta. Prolonged treatment with phorbol ester promoted the inhibitory effects of OA on elastin, as did shorter treatment with IL-1 beta. Moreover, transient transfection studies indicated that OA and IL-1 beta do not act through the same cis-acting element in the elastin promoter. Finally, unlike the transient effects of IL-1 beta, OA induced persistent inhibition of elastin expression by a transcriptional mechanism. Taken together, these data indicate that serine/threonine protein phosphorylation can regulate the amount and composition of extracellular matrix secreted by fibroblasts into the interstitium of the lung.

Animals↗
PubMed1996

Regulation of type I collagen production by insulin and transforming growth factor-beta in human lung fibroblasts.

The effects and interaction of transforming growth factor-beta (TGF-beta) and insulin on collagen production in human fetal lung fibroblasts was examined. Fibroblasts were labeled with [3H]proline and collagen production was analyzed by polyacrylamide gel electrophoresis. The addition of insulin (2 micrograms/ml) increased collagen production 5 fold and TGF-beta (5 ng/ml) increased collagen production 6-fold. The combination of TGF-beta and insulin further increased type I collagen production (12 fold). We found that TGF-beta increased pro-alpha 1 (I) collagen mRNA levels 2-3 fold, insulin increased mRNA levels by less than 2 fold, and the combination stimulated a 3-4 fold increase. In a nuclear run-on assay, we found a 1.7 fold increase in the rate of transcription for the pro-alpha 1 (I) collagen gene in insulin-treated cultures and a 2-fold increase in TGF-treated cultures. In fibroblasts transfected with a plasmid containing 2.4 kb of the 5' flanking sequences of the human pro-alpha 1 (I) collagen gene, TGF-beta stimulated a 2.8 fold increase in promoter activity. In contrast, the addition of insulin stimulated a small increase (less than 2 fold) in the pro-alpha 1 (I) collagen promoter activity when administered alone or in combination with TGF-beta. Insulin prolonged the half-life of pro-alpha 1 (I) collagen mRNA from 9.1 h to 14.3 h as assessed by treatment with actinomycin D. The insulin-induced increase in pro-alpha 1 (I) collagen mRNA was blocked by the presence of cycloheximide indicating a requirement for new protein synthesis. These results show that the combination of TGF-beta and insulin stimulate large increases in type I collagen formation by acting at different sites in the collagen biosynthetic pathway.

Collagen↗
PubMed1996

Differential expression of elastin and alpha 1(I) collagen mRNA in mice with bleomycin-induced pulmonary fibrosis.

Interstitial pulmonary fibrosis is characterized by increased production of connective tissue components, including collagen and elastin. The role of elastin turnover in pulmonary fibrosis is not clear, and it is not known whether elastin and collagen are regulated separately or together during the inflammatory process. Mice with bleomycin-induced pulmonary fibrosis were investigated to determine the temporal and spatial changes in localization of elastin mRNA expression, as well as to compare elastin mRNA expression with that of alpha 1(I) collagen mRNA expression. In control (saline-treated) lungs, elastin mRNA was detected by in situ hybridization in arterial walls. No signal was found in alveolar or airway walls or in pleura; alpha 1(I) collagen mRNA was detected in the tissue underlying the airway epithelium. An increase in elastin mRNA expression in muscular arteries was observed 3 days after bleomycin instillation. Expression was also seen in the adventitia of terminal airways and adjacent small blood vessels. Expression of alpha 1(I) collagen mRNA increased in the tissue underlying the airway epithelium. In the pleura, alpha 1(I) collagen mRNA expression was found, although no pleural thickening was evident. The alpha 1(I) collagen mRNA expression was particularly increased in the adventitia of terminal airways and in associated small blood vessels. Obvious in areas of fibrosis, elastin mRNA expression was occasionally increased in the pleura and airway wall 7 days after bleomycin treatment; alpha 1(I) collagen mRNA expression was generally stronger than elastin mRNA expression in areas of fibrosis and was frequently intense in the adventitia of airways and associated blood vessels. The fibrotic areas showed increased elastin mRNA expression 14 and 30 days after bleomycin treatment. The arteries in fibrotic areas showed normal elastin mRNA levels. In the areas of fibrosis and in the adventitia of airways and adjacent blood vessels, alpha 1(I) collagen mRNA expression was very high. In conclusion, lung elastin biosynthesis is markedly altered by bleomycin treatment. The localization and intensity of elastin mRNA expression is different from the expression of alpha 1(I) collagen mRNA.

Animals↗
PubMed1995

Discordant regulation of transforming growth factor-beta receptors by prostaglandin E2.

The effect of PGE2 on TGF-beta receptor binding was assessed for both signaling (type I and type II) and non-signaling (type III) TGF-beta receptors. We found in cross-linking studies that PGE2 treatment (24 h) decreased binding of TGF-beta to the type I and type II receptors by approx. 50% and markedly increased binding of TGF-beta to type III receptors nearly 10-fold. Northern analyses indicated that PGE2 decreased type I receptor mRNA levels by approximately 30-50%, decreased type II receptor mRNA levels by approximately 60%, and markedly increased type III receptor mRNA levels. Coincubation with cycloheximide partially blocked the PGE2-induced inhibition of type II receptor mRNA. In contrast, cycloheximide minimally affected PGE2-induced type III receptor mRNA levels. Activation of protein kinase C by phorbol esters had no apparent effect on TGF-beta receptor mRNA levels. Our data suggest that alterations in TGF-beta receptor expression could modify the response of tissues to TGF-beta during injury.

Animals↗
PubMed1995

Potential therapeutic initiatives for fibrogenic lung diseases.

Fibrotic process affecting the lung and other tissues is characterized by stimulation of fibroblast proliferation and connective tissue deposition. Conventional therapy consisting of glucocorticoids or cytotoxic agents is usually ineffective in blocking progression of disease. Potential new therapies have emerged from the use of animal models of pulmonary fibrosis and recent advances in the cellular and molecular biology of inflammatory reactions. Such therapies involve the use of substances directed against the action of certain growth factors, cytokines, or oxidants that are elaborated during the fibrotic reaction. In this article, we review possible therapeutic applications of these advances.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts.

We examined alpha 1(I) collagen gene expression in all-trans retinoic acid (RA)-treated human lung fibroblast cultures. RA (10(-5)M) decreased steady-state levels for alpha 1(I) collagen mRNA by at least 75% after 24 h. The inhibition was evident within 8 h after addition of RA, was dose dependent and reversible. Treatment with 9-cis-retinoic acid did not affect alpha 1(I) collagen mRNA levels. RA also inhibited the increases in alpha 1(I) mRNA stimulated by transforming growth factor-beta (TGF-beta). The RA-mediated decrease in alpha 1(I) collagen mRNA was blocked by cycloheximide treatment, suggesting that synthesis of a protein intermediate is required for the inhibition. The RA-induced decrease in alpha 1(I) collagen mRNA levels was not mediated by increases in prostaglandin E2 production. RA decreased alpha 1(I) gene transcription as determined by nuclear run-off assays but did not significantly alter the rate of degradation of the alpha 1(I) transcript as determined by actinomycin D treatment. Studies employing cells stably transfected with constructs containing portions of the alpha 1(I) collagen promoter indicate that the DNA sequences which mediate the inhibitory effect are located within 900 bases from the transcription start site.

Ascorbic Acid↗
PubMed1994

Structure and expression of the promoter for the human type II transforming growth factor-beta receptor.

The type II TGF-beta receptor is a serine/threonine kinase whose expression is essential for the action of TGF-beta. In this paper, we describe the cloning and expression of the human type II TGF-beta promoter. DNA sequence analysis indicates that the region near the transcription initiation site lacks a TATA and CAAT box but contains Sp1 binding sites. A similar promoter type has been identified in the 5' flanking regions of genes that code for certain other growth factor receptors. Transfection of the promoter (888 bp fragment) mediated transcriptional activity in bovine vascular smooth muscle cells and human lung fibroblasts.

Animals↗
PubMed1994

Type I collagen formation in rat type II alveolar cells immortalised by viral gene products.

BACKGROUND: Alveolar type II (T2) cells synthesise matrix proteins such as type IV collagen and fibronectin. In contrast, a fetal rat T2 cell line has been shown to synthesise type I and III collagen as well as type IV collagen. To study regulation of collagen production in T2 cells, neonatal T2 cells immortalised by adenoviral 12SE1A gene transfer were used. It was previously reported that this immortalised cell line (E1A-T2) retains epithelial features such as tight junctions and cytokeratins but also expresses mesenchymal features such as vimentin. METHODS: Collagen production was examined in E1A-T2 and primary neonatal T2 cells using polyacrylamide gel electrophoresis. Electron microscopy was used to examine collagen deposition in E1A-T2 cell culture. To define the mechanism by which alpha 1(I) type I collagen gene expression was activated in E1A-T2 cells, a deletional analysis of alpha 1(I) promoter constructs linked to the bacterial chloramphenicol acetyltransferase gene was performed. RESULTS: E1A-T2 cells produced large amounts of type I collagen with a predominance of alpha 1(I) homotrimers; alpha 2(I) peptides were detected only in the cell layer. In contrast, primary neonatal rat T2 cell cultures produced a trace amount of type I collagen. Production of alpha 1(I) peptide chains (per microgram DNA) in E1A-T2 cell cultures was 30 times higher than that observed in primary neonatal T2 cell cultures. Electron microscopy showed deposition of type I collagen fibrils in the extracellular matrix of E1A-T2 cell cultures. Transfection studies suggested at least two cis-acting elements which mediate increased alpha 1(I) gene expression in E1A-T2 cells. CONCLUSIONS: These studies indicate that the E1A-T2 cell line may be useful for studying type I collagen gene regulation in alveolar T2 cells. These findings also raise the possibility that viral activation of type I collagen genes in alveolar epithelium may be involved in certain forms of pulmonary fibrosis.

Animals↗
PubMed1994

Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-beta 1 and prostaglandin E2.

The regulation of lysyl oxidase produced by cultured, lipid-enriched, neonatal rat lung fibroblasts was explored. The presence of 40 pM of transforming growth factor-beta 1 (TGF-beta 1) in overnight cultures increased levels of enzyme secreted into the medium by 1.6-fold while steady-state levels of lysyl oxidase mRNA increased similarly. In contrast, incubation of these cultures with 100 nM of prostaglandin E2 (PGE2) reduced enzyme activity levels by 40 to 50% although steady-state mRNA was not changed. Consistent with the effect of PGE2, the presence of indomethacin stimulated levels of secreted enzyme activity. When present in cultures simultaneously with TGF-beta 1, PGE2 prevented the stimulation beyond control levels seen with TGF-beta 1 alone. Densitometry of protein bands immunoprecipitated by antibody to lysyl oxidase indicated that the degree of conversion of the 50 kD proenzyme to the 29 kD enzyme was not significantly altered by TGF-beta 1 or PGE2. However, the net accumulation of all forms of lysyl oxidase protein was increased by TGF-beta 1 and decreased by PGE2. These results indicate that TGF-beta 1 and specific prostaglandin(s) exert opposing effects on the expression of lysyl oxidase in these lung fibroblasts.

Animals↗
PubMed1993

Effects of transforming growth factor beta and interleukin-1 beta on expression of cyclooxygenase 1 and 2 and phospholipase A2 mRNA in lung fibroblasts and endothelial cells in culture.

Experiments were conducted to determine the roles of the rate limiting enzymes, cyclooxygenase 1 and 2 (COX1 and COX2) and cytoplasmic phospholipase A2 (PLA2), in transforming growth factor beta (TGF-beta) and interleukin-1 (IL-1 beta) activated prostaglandin synthesis. Results show that TGF-beta increases steady state levels of COX1 mRNA in both human embryo lung fibroblasts (IMR90) and calf pulmonary artery endothelial cells (BPAEC). Temporal experiments show that TGF-beta increases, within 2hrs, a 5.5kb COX1 in IMR90 and the 2.7kb COX1 mRNA in BPAEC. IL-1 beta increases COX1 mRNA only in IMR-90, not BPAEC. COX2 mRNA, under basal conditions, is not detected in BPAEC and is expressed only marginally in IMR90. TGF-beta or IL-1 beta have no effect on expression of COX2 gene in either cell type. IL-1 beta increases steady state levels of PLA2 mRNA in both IMR90 and BPAEC while TGF-beta increases expression of the PLA2 gene only in BPAEC. Time experiments with TGF-beta show induction of PLA2 mRNA within 1hr, peaking at 4hrs. PG synthesis in response to the cytokines was determined in IMR90 and BPAEC to further assess the significance of the above results. TGF-beta increases the synthesis of prostacyclin in BPAEC in a time related fashion peaking at 8hrs at 13 fold above basal. To focus on the action of COX1 and bypass the action of PLA2, exogenous arachidonic acid was used as substrate for PG synthesis. In these experiments IL-1 beta increases PGE2 synthesis 8 fold in IMR90 while IL-1 beta and TGF-beta added simultaneously increases PGE2 synthesis 25 fold. These results in sum illustrate that the cytokines, TGF-beta and IL-1 beta, regulate both COX1 and PLA2 mRNA levels. Furthermore, this regulation appears coordinated to bring about elevation of prostaglandin synthesis.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

The effect of retinoic acid on amino acid uptake and protein synthesis by lung fibroblasts.

The effect of retinoic acid (RA) on the uptake and utilization of extracellular amino acids by fetal lung fibroblasts was examined. RA decreased the incorporation of [3H]proline into collagen and other proteins. The effect was maximal at a RA concentration of 10(-5) M; smaller decreases were observed at a RA concentration of 10(-6) M. This decrease in collagen formation was associated with a large decrease in intracellular [3H] proline. The decrease in intracellular [3H]proline was first observed at 2 h following the addition of RA to cell cultures. Transport studies employing radiolabeled amino acids revealed that RA decreased the uptake of proline, 2-aminoisobutyric acid, and 2-(methylamino)isobutyric acid but not leucine or methionine. Kinetic analysis of 2-aminoisobutyric acid uptake indicated that this effect was mediated primarily by an increase in apparent Km, with a lesser decrease in Vmax, RA-induced inhibition of proline uptake was not abolished by the presence of cycloheximide nor by pretreatment with indomethacin. Na+,K(+)-ATPase activity was not affected by RA treatment. These results suggest that RA modulates protein production in fibroblasts by altering the function of the Na(+)-dependent A transport system for amino acid uptake.

Amino Acids↗
PubMed1993

Regulation of the alpha 1(I) collagen promoter via a transforming growth factor-beta activation element.

A transforming growth factor-beta (TGF-beta) activating element (TAE), with a nuclear factor-1 (NF-1)-like sequence, was previously located 1.6 kilobases upstream from the transcription start site in the alpha 1(I) collagen promoter (Ritzenthaler, J. D., Goldstein, R. H., Fine, A., Lichtler, A., Rowe, D. W., and Smith, B. D. (1991) Biochem. J. 280, 157-162). Double-stranded TAE, but not NF-1 consensus sequences, abrogated TGF-beta stimulation of co-transfected collagen promoter-chloramphenicol acetyltransferase constructs. Mutations in non-NF-1 binding sites, located by methylation interference, eliminated activity of the TAE oligonucleotide. However, TAE sequences failed to bind in vitro expressed NF-1 protein, to compete for NF-1-binding proteins, and to bind with protein which reacts with antibodies to NF-1 family of proteins. Within the TAE there was an activator protein 2 (AP-2) binding site. Although AP-2 protein bound to TAE, antibodies to AP-2 did not react with nuclear protein-TAE complexes. TAE bound to a 34,000-Da protein on Southwestern analysis. However, the UV-cross-linked TAE-nuclear protein complex was 82,000 Da. Finally, a dose-response study demonstrated that TGF-beta increased TAE nuclear binding proteins at lower doses with a different response curve than NF-1 nuclear binding proteins. Taken together these data demonstrated that TGF-beta functions in human lung fibroblasts to activate collagen transcription through TAE sites by protein complexes independent of NF-1 or AP-2 protein.

Base Sequence↗