Biomedical subjects

B C Trapnell

Publications and source records attributed to B C Trapnell.

At least 55 records · Page 3Linked to original sources

PubMed1992

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium.

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.

Adenoviruses, Human↗

PubMed1992

Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents that modulate intracellular divalent cations.

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Calcimycin↗

PubMed1992

Extensive posttranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts of the cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis.

Cystic fibrosis (CF) is a recessive hereditary disorder, requiring both parental cystic fibrosis conductance transmembrane regulator (CFTR) genes to carry mutations for clinical disease to manifest, i.e., only 50% of normal CFTR gene expression is required to maintain a normal phenotype. To help define the minimum amount of normal CFTR gene expression necessary to maintain normalcy, we have capitalized on our prior observation (Chu, C.-S., B. C. Trapnell, J. J. Murtagh, Jr., J. Moss, W. Dalemans, S. Jallat, A. Mercenier, A. Pavirani, J.-P. Lecocq, G. R. Cutting, et al. 1991. EMBO [Eur. Mol. Biol. Organ] J. 10:1355-1363) that normal individuals can have up to 66% of bronchial CFTR mRNA transcripts that are missing exon 9, a region representing 21% of the sequence coding for the critical nucleotide (ATP)-binding fold 1 (NBF1) of the predicted CFTR protein. The study population included 78 individuals with no prior diagnosis of CF. Evaluation of bronchial epithelial cells (obtained by bronchoscopy) revealed that exon 9 was variably deleted in all individuals. Remarkably, there were four individuals, all greater than or equal to 35 yr, in whom bronchial epithelial cells exhibited 73, 89, 90, and 92% CFTR transcripts with inframe deletion of exon 9, respectively, despite normal sweat Cl- and no clinical manifestation of CF. In the context that only 8% or less of bronchial CFTR transcripts need exon 9 to maintain normal airway function, these observations strongly suggest that either exon 9 is not necessary for CFTR structure and/or function or that only a very small fraction of bronchial epithelial cells need to express normal CFTR mRNA transcripts with exon 9 to perform the function of CFTR sufficient to maintain a normal phenotype in vivo.

Adult↗

PubMed1991

Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.

Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL-60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts.

Base Sequence↗

PubMed1991

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis.

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called "delta Phe508," a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, we evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-delta Phe508 heterozygotes, and delta Phe508 homozygotes to determine if the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium, to what extent they are expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and delta Phe508 CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells at approximately 1-2 copies per cell, more than 100-fold greater than in pharyngeal epithelium. Importantly, allele-specific hybridization studies demonstrated that the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium in similar amounts.

Base Sequence↗

PubMed1991

Down-regulation of cystic fibrosis gene mRNA transcript levels and induction of the cystic fibrosis chloride secretory phenotype in epithelial cells by phorbol ester.

To evaluate the hypothesis that phorbol myristate acetate (PMA) might modulate the expression of the cystic fibrosis (CF) gene in epithelial cells, we examined the effect of PMA on CF mRNA levels and regulation of Cl- secretion. Strikingly, PMA down-regulated CF mRNA transcript numbers in a dose- and time-dependent manner. Importantly, in parallel with the reduction of CF mRNA levels, PMA-treated cells were unable to up-regulate Cl- secretion in a normal fashion in response to forskolin, an effect which was also dose- and time-dependent. Thus, PMA is capable of modulating expression of the CF gene and induces T84 cells to adopt the "CF phenotype" in regard to regulation of Cl- ion transport.

Blotting, Northern↗

PubMed1991

The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin.

Evaluation of the expression of the cystic fibrosis (CF) gene in human epithelial cell lines demonstrated active, but low level, transcription of the gene. Analysis of 3.8 kilobases of genomic sequences 5' to exon 1 of the CF gene demonstrated no TATA promoter element, but a high G + C content, multiple transcription start sites, and several potential Sp1 binding sites. Fragments of 5'-flanking sequences from 2.2 kilobases to as small as 102 base pairs 5' to the major transcription start site supported constitutive reporter gene expression in epithelial cells, but at low levels, and independent of the length of the 5' fragment. CF gene transcription was down-regulated by phorbol myristate acetate. Importantly, evaluation of freshly isolated normal human bronchial cells also demonstrated CF gene transcription at a relatively low rate. Together, these observations suggest that although the normal CF gene promoter has characteristics of a "housekeeping"-type gene, and the gene is expressed at low levels in cells of organs that manifest the clinical disorder "cystic fibrosis," its expression can be modulated transcriptionally, implying a possible therapeutic approach for the disease.

Base Sequence↗

PubMed1991

Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium.

The predicted protein domains coded by exons 9-12 and 19-23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide-binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9- mRNA transcripts). In most (9 of 12), the exon 9- transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9- transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re-evaluation of the same individuals 2-4 months later showed the same proportions of exon 9- transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon-intron junctions relevant to exon 9 deletion (exon 8-intron 8, intron 8-exon 9, exon 9-intron 9, and intron 9-exon 10) were identical except for the intron 8-exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9- transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype.

Base Sequence↗

PubMed1991

Expression of the secretory leukoprotease inhibitor gene in epithelial cells.

The secretory leukoprotease inhibitor (SLPI) gene codes for a 12-kD protein that within the lung protects the airway epithelium from neutrophil elastase. Screening of 228 alleles in 114 individuals for sequence differences by RNase protection of genomic DNA revealed no detectable polymorphisms in SLPI gene exons II-IV. SLPI gene expression in the lung was demonstrated by identifying SLPI mRNA transcripts in bronchial epithelial cells freshly isolated from normals. Cell lines derived from mucosal surfaces (HS-24 bronchial squamous cell carcinoma, HeLa cervical carcinoma) actively transcribe the SLPI gene and contain SLPI mRNA transcripts, while lung fibroblasts demonstrate no evidence of SLPI gene expression. SLPI mRNA transcripts appear to be relatively stable, with mRNA levels only mildly affected by inhibition of RNA synthesis. Chromatin DNA of HS-24 cells demonstrates two DNase I hypersensitivity sites within the 5' flanking region of exon I of the SLPI gene, whereas fibroblast chromatin has no DNase I accessible sites in the same region. Further analysis of the 5' flanking region demonstrated two contiguous transcription start sites, CAAT and TATA boxes, and several potential regions of known DNA binding proteins. Overall, the SLPI gene appears to be a relatively nonpolymorphic, stable gene that is constitutively expressed at specific tissue sites, but has the potential to be modulated at both the transcriptional and posttranscriptional levels.

Base Sequence↗

PubMed1991

Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas.

Adolescent↗

PubMed1990

Biased accumulation of T lymphocytes with "memory"-type CD45 leukocyte common antigen gene expression on the epithelial surface of the human lung.

Expression of alternatively spliced products of the CD45 leukocyte common antigen gene identifies two populations of blood T cells: "naive" T cells (containing CD45R-IV mRNA transcripts, CD45 220, 205 kD surface proteins detected with antibody 2H4) that respond poorly to recall antigens, and "memory" T cells (containing CD45R-0 mRNA transcripts, expressing CD45 180 kD protein, detected with antibody UCHL1) that respond promptly to recall antigens. While blood contains approximately equal numbers of "naive" and "memory" T cells, it is known that UCHL1+ "memory" T cells accumulate at sites of chronic inflammation. To test the concept that "memory" T cells are a feature of the T lymphocyte populations present in tissues chronically exposed to antigens in normals as well as in individuals with chronic inflammation, we evaluated T lymphocytes obtained from blood and the epithelial surface of the lower respiratory tract of normal individuals for the expression of specific CD45 surface protein isoforms and corresponding mRNA transcripts. Flow cytometric analysis of CD45 220, 205, and 180 kD surface proteins demonstrated that lung T cells of normals are dominated by UCHL1+ "memory" cells (86 +/- 2%) while autologous blood T cells have equal proportions of "memory" UCHL1+ and "naive" 2H4+ T cells. In addition, polymerase chain reaction analysis of CD45 mRNA transcripts revealed that the lung cells expressed CD45R-0 mRNA transcripts but 17-fold fewer CD45R-IV mRNA transcripts than autologous blood T cells (p less than 0.01). The pattern of lung T cells being dominated by CD45R-0 mRNA+, UCHL1+ "memory" T cells was also observed in individuals with chronic beryllium disease, an example of a chronic inflammatory disease in which antigen-specific T cells accumulate on the pulmonary epithelial surface. Like the normals, the lung T cells of the beryllium disease patients were dominated by CD45R-0 mRNA transcript+, UCHL1+, T cells. However, on a quantitative basis, the beryllium patients contained far greater numbers of T cells, i.e., the T cell populations on the surface of the normal and inflamed lung are similar in character ("memory" T cells) but differ in numbers (there are far more in the chronic inflammatory state). Thus, T cell populations on the epithelial surface of the normal lung likely reflect the chronic exposure to a diverse set of antigens, with a pattern that is qualitatively similar to that observed among T cells accumulating in response to a single antigen.

Adult↗

PubMed1990

Regulation of insulin-like growth factor I gene expression in the human macrophage-like cell line U937.

Activated macrophages release tissue forms of insulin-like growth factor I (IGF-I), 20-25-kD products of the IGF-I gene, thus providing an extracellular growth and differentiation signal at sites of inflammation. To examine the control of IGF-I gene expression in mononuclear phagocytes, the human macrophage-like cell line U937 was evaluated at rest and after surface activation with phorbol myristate acetate (PMA) or Ca2+ ionophore. Northern analysis and RNAse protection analysis with 32P-labeled IGF-I-specific probes demonstrated that the IGF-I mRNA transcripts of resting U937 cells were similar in size and amount to those of resting human alveolar macrophages, mononuclear phagocytes known to express the IGF-I gene. Nuclear run-off assays demonstrated that surface activation of U937 cells increased the transcription rate of the IGF-I gene four- to fivefold, a process that was inhibited by cycloheximide, suggesting that active protein synthesis was involved in the activation pathway. Despite this, cytoplasmic IGF-I mRNA levels after surface activation declined markedly, a process blocked by a protein kinase C inhibitor (for PMA activation) or a calmodulin antagonist (for Ca2+ ionophore activation). Like the increased transcription of the IGF-I gene, modulation of IGF-I mRNA transcript levels required active protein synthesis; in the presence of cycloheximide constitutive IGF-I mRNA levels increased and surface activation no longer caused a decrease in transcript number. Interestingly, surface activation caused a rapid release of IGF-I, even in the presence of a protein synthesis inhibitor, suggesting that mononuclear phagocytes have a preformed, stored, releasable pool of IGF-I. Together these observations demonstrate that IGF-I gene expression is complex and probably involves control of transcription rate, cytoplasmic mRNA levels possibly mediated through protein kinase C, calcium influx and calmodulin, and finally, release of preformed IGF-I from a storage pool.

Calcimycin↗

PubMed1990

Upregulation of platelet-derived growth factor-A and -B gene expression in alveolar macrophages of individuals with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of alveolar macrophages spontaneously releasing exaggerated amounts of the potent mesenchymal cell growth factor platelet-derived growth factor (PDGF). To evaluate the relative contribution of the two PDGF genes to this process, PDGF-A and -B gene transcription rates and mRNA levels were examined in normal and IPF alveolar macrophages. While normal alveolar macrophages constitutively transcribe both PDGF-A and PDGF-B genes, LPS stimulation increases the transcription of both genes more than threefold. Importantly, IPF alveolar macrophages spontaneously transcribe both genes at a rate similar to that observed for normal macrophages after in vitro stimulation. Consistent with the transcription data, normal macrophages contain mRNA for both PDGF-A and -B, but PDGF-B mRNA is 10-fold more abundant. Strikingly, in IPF, both PDGF-A and -B mRNA levels were markedly increased, with persistence of the 10-fold dominance of PDGF-B mRNA. Thus, the exaggerated release of PDGF by IPF alveolar macrophages is likely modulated by upregulated PDGF gene transcription rates and concomitantly increased mRNA levels and the persistent 10-fold excess of B greater than A PDGF mRNA suggests that the PDGF released by alveolar macrophages is likely mostly of the potent B-chain homodimeric form.

Blotting, Northern↗

PubMed1988

Alveolar macrophages release an insulin-like growth factor I-type molecule.

Human alveolar macrophages, when activated, release a progression-type growth factor for fibroblasts that signals "competent" fibroblasts to replicate. The present study demonstrates that this growth activity is an insulin-like growth factor I (IGF-I)-type molecule. Partial purification of medium conditioned by activated alveolar macrophages using ion exchange and gel filtration chromatography revealed an IGF-I molecule as detected by an anti-IGF-I polyclonal antibody and that the specific activity of the progression-type growth activity tracked with the amount of IGF-I present. In a serum-free complementation test, the increase in fibroblast proliferation by alveolar macrophage IGF-I was reduced in a dose-response manner with an anti-IGF-I monoclonal antibody. The alveolar macrophage IGF-I displaced 125I-IGF-I from its receptor in a binding assay utilizing human lung fibroblasts and it stimulated type I IGF receptors purified from human lung fibroblasts to phosphorylate a tyrosine-containing artificial substrate. In contrast to the 7.6-kD serum IGF-I, gel chromatography revealed that the alveolar macrophage IGF-I had an apparent molecular mass of 26 kD, similar to other tissue IGF-Is. Alveolar macrophages expressed IGF-I mRNA transcripts as detected by solution hybridization using a 32P-labeled riboprobe complementary to exons I-II-III of the IGF-I gene. In the context of the known functions of the family of IGF-I molecules in cell growth, IGF-I released by activated alveolar macrophages may play a role in acute and chronic inflammatory disorders.

Binding, Competitive↗

PubMed1986

Metoclopramide suppositories in the treatment of diabetic gastroparesis.

Gastroparesis diabeticorum is a common complication that develops in patients with diabetes mellitus. Although the pathogenesis remains unclear, the clinical symptoms of nausea, vomiting, and gastric dilatation frequently respond to metoclopramide hydrochloride, an agent that stimulates gastric emptying in addition to acting centrally as an antiemetic. Occasionally, patients are encountered whose severe gastroparesis is unresponsive to oral metoclopramide and who require intravenous therapy or drainage procedures (eg, pyloroplasty or gastrojejunostomy). Rectal administration of metoclopramide successfully controlled the clinical symptoms of gastroparesis diabeticorum in an outpatient after failure of oral dosing, thus avoiding the need for intravenous therapy. Gastric emptying studies and serum metoclopramide levels following a 25-mg rectal dose of metoclopramide hydrochloride verified the efficacy of therapy.

Adult↗

PubMed1982

Copy number of a human type I alpha 2 collagen gene.

HpCl, a Charon 4A bacteriophage containing a 16.3-kilobase insert of human genomic DNA, has been identified as representing the 3' portion of the human alpha 2(I) collagen gene by hybrid selected translation and cross-reactivity with characterized sheep and chick collagen genes. Evaluation of HpCl by restriction endonuclease mapping and alpha 2(I) mRNA hybridization demonstrated that the alpha 2(I) mRNA coding regions are separated by noncoding regions including one of 2.7 kilobase pairs near the 3' end of the mRNA coding region. In addition, a 1.8-kilobase XbaI-BamHI fragment containing the 2' a 1.8-kilobase XbaI-BamHI fragment containing the 3' end of the alpha 2(I) mRNA coding region includes a region of DNA that is repeated many times throughout the human genome. Quantification of alpha 2(I) gene number, using the technique of DNA dot hybridization, with two EcoRI fragments from HpClo as probes, demonstrated that HpCl is represented once in the human genome. In this context, it is unlikely that human alpha 2(I) collagen chain production is modulated by differential transcription of multiple alpha 2(I) genes.

Animals↗

PubMed1981

Procollagen messenger RNA levels and activity and collagen synthesis during the fetal development of sheep lung, tendon, and skin.

The rates of type I collagen synthesis in sheep lung, tendon, and skin were evaluated during the latter half of fetal development and compared with the levels of type I procollagen mRNA, quantified by molecular hybridization with a type I procollagen specific complementary DNA, and with the activity of total procollagen mRNA measured by in vitro cell-free translation. In the lung and tendon, the levels of type I procollagen mRNA and activity of total procollagen mRNA parallel collagen synthesis during development. In the skin, however, type I collagen synthesis declines sharply during fetal development, but both type I procollagen mRNA levels and total procollagen mRNA activity remain at the high levels of early development. These observations suggest that in developing lung and tendon, type I procollagen mRNA levels are likely the major determinants of the levels of type I collagen synthesis. In contrast, the dichotomy between type I procollagen mRNA levels and rates of type I collagen synthesis in the developing sheep skin suggest the skin utilizes mechanisms in addition to mRNA levels to modulate expression of the type I collagen gene.

Animals↗

PubMed1981

Procollagen production and procollagen messenger RNA levels and activity in human lung fibroblasts during periods of rapid and stationary growth.

The production of procollagen molecules by human diploid fetal lung fibroblasts (HFL-1 cells) remains constant in both rapid and stationary growth phases. However, log phase cells degrade 3-fold more newly synthesized collagen inside the cell prior to secretion than do stationary phase cells. Procollagen mRNA levels, measured by hybridization with a type I procollagen mRNA-specific complementary DNA, are approximately 2-fold higher in confluent cells than in log phase cells. There are no significant differences in the ability of either log phase or confluent HFL-1 cell procollagen mRNA to be translated in an in vitro cell-free translation system. Therefore, the ability of HFL-1 cells to maintain constant collagen production irrespective of the growth status of the cells results from the combined action of a number of regulatory mechanisms, including changes in procollagen mRNA levels, the utilization of procollagen mRNA, and intracellular procollagen degradation.

Cell Division↗