Mouse alpha 1 type V collagen gene maps to the [A2-B] region of chromosome 2.
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Animals↗
Biomedical subjects
G Karsenty
Publications and source records attributed to G Karsenty.
Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.
We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.
Animals↗
Purification and functional characterization of a DNA-binding protein that interacts with a negative element in the mouse alpha 1(I) collagen promoter.
In an effort to understand the regulation of expression of the mouse type I collagen genes, we have purified to homogeneity a transcription factor, called inhibitory factor 2 (IF-2), that binds to the mouse alpha 1(I) collagen promoter upstream of the proximal CCAAT motif. IF-2 was purified to homogeneity from mouse lymphocyte nuclear extracts by ion-exchange chromatography and two different steps of DNA affinity chromatography, one using the wild-type IF-2-binding site as affinity ligand and one using a mutated IF-2-binding site that has a higher affinity for the factor. Renaturation of active DNA-binding proteins purified through several chromatography steps from sodium dodecyl sulfate-polyacrylamide gels identified two polypeptides of 120 and 100 kDa, respectively, capable of binding specifically to an IF-2-binding site. DNA transfection experiments of NIH 3T3 fibroblasts using an alpha 1(I) promoter-CAT chimeric gene in which mutations were introduced that either improved or decreased the binding affinity of IF-2 for its recognition site, strongly suggest that IF-2 acts as a transcriptional inhibitor of the mouse alpha 1(I) collagen gene. DNA-binding studies with similar concentrations of purified IF-2 and CBF (CCAAT-binding factor) indicate that CBF inhibits binding of IF-2, whereas IF-2 inhibits CBF binding more weakly.
Animals↗
An intron binding protein is required for transformation ability of p53.
Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.
Animals↗
Conservation of binding sites for regulatory factors in the coordinately expressed alpha 1 (I) and alpha 2 (I) collagen promoters.
To examine possible mechanisms for the coordinate control of the alpha 1 (I) and alpha 2 (I) collagen genes, we have searched for DNA binding factors that are common to both genes. We have recently identified in the proximal part of the alpha 1 (I) promoter a functional binding site for CBF, a heteromeric transcriptional activator which binds to certain CCAAT sequences, and also functional binding sites for two different transcriptional repressors, designated IF1 and IF2. CBF was previously also shown to bind and activate the alpha 2(I) collagen promoter. We now present evidence that a factor with similar binding characteristics as IF1 binds to the alpha 2(I) promoter at approximately the same distances from the start site of transcription as in the alpha 1(I) collagen promoter. A three bp substitution mutation in the IF1 binding site which abolishes IF1 binding increases the activity of the alpha 2(I) promoter 4-fold as with the alpha 1 (I) promoter. We propose that the coordinate regulation of these two genes is at least in part mediated by these common elements.
Animals↗
A fibroblast-specific factor binds to an upstream negative control element in the promoter of the mouse alpha 1(I) collagen gene.
We have identified, in nuclear extracts of NIH-3T3 fibroblasts, a factor which binds a sequence between -339 and -361 in the mouse alpha 1(I) collagen promoter. A 3-base pair substitution mutation introduced in this promoter element, which abolishes the binding of the factor, increases the activity of the promoter 4-fold in DNA transfection experiments. This suggests that this element is a negative regulator of transcription. The factor which binds to the -339 to -361 sequence is present in type I collagen-producing cell lines but not in a variety of other cell types, suggesting that it is involved in the tissue-specific regulation of the alpha 1(I) gene.
Animals↗
Transcriptional control mechanisms for the expression of type I collagen genes.
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Animals↗
A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos.
The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at -70 bp to -75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at -721 bp to -726 bp. The proximal 'G-string' element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.
Animals↗
Two different negative and one positive regulatory factors interact with a short promoter segment of the alpha 1 (I) collagen gene.
Type I collagen, a heterotrimeric protein composed of two alpha 1 chains and one alpha 2 chain, is a major specialized biosynthetic product of fibroblastic cells. We performed a functional dissection of a mouse alpha 1 (I) collagen promoter segment (between -222 and -80) that displays strong activity in vitro and in DNA transfection experiments. Four binding sites for factors present in nuclear extracts of NIH-3T3 fibroblasts were identified in this promoter segment. One factor, which has the same binding characteristics as CBF, a heterodimeric CCAAT binding factor that also binds and activates the coordinately expressed alpha 2(I) collagen promoter, interacts with the proximal of two CCAAT motifs. A second factor, designated IF1, binds to two more upstream, adjacent sites (-190 to -170 and -160 to -130). A third factor, designated IF2, makes contact with several G residues in the distal unit of a 12-base pair exact G-rich repeat that brackets the proximal CCAAT motif. Binding studies with mutant oligonucleotides and experiments with purified CBF indicate that the binding of IF2 is inhibited by CBF. IF2 is a metalloprotein that requires zinc cations for efficient binding to its recognition site. DNA transfection experiments using point mutations or small substitution mutations that abolish binding of the transacting factors to their cognate elements were performed. A mutation in the proximal IF1 binding site increases promoter activity 4-fold, a mutation in the IF2 binding site increases this activity 10-fold and a mutation in the CBF binding site decreases this activity 4-5-fold. This suggests that IF1 and IF2 act as transcriptional inhibitors whereas CBF acts as an activator of the alpha 1(I) collagen promoter. We propose that mutually competitive binding of IF2 and CBF could play a role in the control of the alpha 1(I) collagen promoter.
Animals↗
Transcriptional mechanisms controlling types I and III collagen genes.
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Animals↗
Control of type I collagen genes in scleroderma and normal fibroblasts.
Increased gene transcription is a mechanism responsible for the exaggerated accumulation of type I and III collagens in scleroderma and other fibrotic syndromes. We review recent studies on the mechanisms that control transcription of the alpha 1 (I) and alpha 2 (I) collagen genes. Several specific DNA binding proteins have been identified which either activate or inhibit transcription of these genes. It is probable that the complexity of transcription factors that control the type I collagen genes corresponds to a multiplicity of cytokines and other hormonal effectors, which influence the expression of these genes by binding to specific receptors and by activating intracellular signaling pathways.
Collagen↗
Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter.
Point mutations and small substitution mutations were introduced in three different promoter segments of the mouse alpha 2(I) collagen gene that are binding sites for specific DNA-binding proteins. The first of these promoter elements contains a CCAAT motif between -80 and -84 and binds a novel factor consisting of two different components which are both needed for DNA binding (Hatamochi, A., Golumbek, P.T., Van Schaftingen, E., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 5940-5947). Four different point mutations in the CCAAT motif, which strongly inhibit binding of this heterodimeric CCAAT-binding factor, decrease the activity of the promoter at least 8-fold when assayed by DNA transfection of NIH 3T3 fibroblasts. The second cis-acting element is located around -250. Two mutations in this element, one a 3-base pair (bp), another a 4-bp substitution mutation, decrease the activity of the promoter in DNA transfection experiments by about 8- and 12-fold, respectively. The third element is located between -315 and -295 and binds nuclear factor 1 (NF1). Both 1- and 2-bp mutations which strongly inhibit binding of NF1 to this site decrease the activity of the promoter 8-10-fold in DNA transfection assays whereas another mutation in this site shows less inhibition in the activity of the promoter. Transfer of this NF1-binding site 5' to the SV40 early promoter increases the activity of this promoter more than 10-fold. These results indicate that the integrity of each of these three cis-acting elements is required for optimal activity of the alpha 2(I) collagen promoter and suggest a model whereby the factors which bind to these sequences cooperate in the formation of a transcription initiation complex.
Animals↗
Selective activation of transcription by a novel CCAAT binding factor.
A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the alpha 2(I) collagen, the alpha 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.
Animals↗
A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta.
Transforming growth factor-beta (TGF-beta) increases the steady-state RNA levels of several fibroblast extracellular matrix proteins. Using DNA transfection, we show that TGF-beta stimulates the activity of the mouse alpha 2(l) collagen promoter 5- to 10-fold in mouse NIH 3T3 and rat osteosarcoma cells. Deletion analysis indicates that a segment of this promoter between -350 and -300, overlapping a nuclear factor 1 (NF1) binding site, is needed for TGF-beta stimulation. A 3 bp substitution mutation abolishing NF1 binding to this site inhibits TGF-beta activation. Insertion of this NF1 binding site 5' to the SV40 early promoter makes the promoter TGF-beta inducible, but the 3 bp substitution does not. Similarly, when the NF1 binding site at the replication origin of adenovirus 2 and 5 is inserted 5' to the SV40 promoter, the promoter responds to TGF-beta. Therefore an NF1 binding site mediates the transcriptional activation of the mouse alpha 2(l) collagen promoter by TGF-beta.
Animals↗
Thyrotropin induces growth and iodothyronine production in a human thyroid cell line without affecting adenosine 3',5'-monophosphate production.
Using a recently described cell line derived from the fusion of human thyroid and human myeloma cells, we studied the effects of TSH on cell growth, iodide organification, and cAMP production. Although these cells grow in the absence of TSH, when incubated for 2 days in serum-free medium containing purified human or bovine TSH, there was a significant increase in cellular DNA content. The stimulatory effect was observed at concentrations as low as 0.5 microU/ml, which produced a significant increase in DNA content, and was maximal with 5 microU/ml. This effect was still present after 6 days of incubation. Electron microscopy performed by an unbiased observer on cells incubated in the presence of TSH showed an increase in the calculated size of the cells and the nucleus which was significant at 0.1 microU/ml and maximal at 1 microU/ml. Stimulation of 125I organification and hormone production was observed at TSH concentrations as low as 1 microU/ml and was maximal at 10 microU/ml. However, neither TSH (1-50 microU/ml) nor forskolin (10(-6) M) stimulated cAMP production. These data suggest that these cells lack a functional adenylate cyclase pathway and that growth and iodide organification are mediated by other second messenger systems. Such a cell line could yield new insights into the mechanisms of TSH action and may provide a sensitive bioassay for TSH and other thyrotropic factors.
Cell Division↗
Hashimoto's thyroiditis with a monoclonal antithyroglobulin autoantibody: disappearance of the monoclonal antibody after thyroidectomy.
A 46-yr-old man had typical Hashimoto's thyroiditis (HT) and a serum monoclonal immunoglobulin (MIgG kappa). There was no evidence of disseminated lymphoid malignancy. Because of rapid enlargement of the goiter with pressure symptoms, a total thyroidectomy was performed. The surgery was followed by the complete disappearance of the MIgG kappa paralleled by the disappearance of serum antithyroglobulin autoantibodies. Thyroid sections demonstrated HT. Direct immunofluorescence assay showed diffuse infiltration by lymphocytes and plasma cells that were mainly IgG kappa positive. Moreover, the serum MIgG kappa had antithyroglobulin activity, as demonstrated by two different methods, namely labeling of focused serum proteins with radioiodinated human thyroglobulin and immunoadsorption of the monoclonal IgG on a human thyroglobulin affinity column. These results demonstrate that a monoclonal antithyroglobulin autoantibody was produced within the thyroid in a patient with HT. As the production of a MIg is regarded as the last stage preceding the malignant transformation of normal B lymphocytes, this report strongly suggests that a B lymphoma can directly emerge from the intrathyroidal lymphocytes involved in the pathogenesis of Hashimoto's thyroiditis.
Antibodies, Monoclonal↗
Cyclosporin increases the rate and length of remissions in insulin-dependent diabetes of recent onset. Results of a multicentre double-blind trial.
In a double-blind trial 122 patients aged 15-40 years with insulin-dependent diabetes of recent onset were randomly assigned to cyclosporin 7.5 mg/kg per day or placebo. At the sixth month 25.4% of the cyclosporin group and 18.6% of the placebo group were in complete remission (not a significant difference). Treatment was continued in those patients with complete or partial remission (insulin requirement less than 0.25 U/kg per day) and 106 patients were followed to nine months, at which stage 24.1% of the original cyclosporin group and 5.8% of the original placebo group were in complete remission (p less than 0.01). For those patients whose whole-blood trough cyclosporin levels in the first three months averaged 300 ng/ml or more, the rates of complete remission at six and nine months were 37.5% and 37%. The rates of partial remission were also higher in the cyclosporin group and at six months the rate of complete or partial remission was 46% in the whole cyclosporin group and 65.6% in those with an average blood level exceeding 300 ng/ml in the first three months, versus 28.8% in the placebo group. The principal side-effect of cyclosporin was a modest and reversible increase in plasma creatinine. These results indicate that cyclosporin promotes the remission of type I diabetes and suggest the need for new controlled protocols aimed at evaluating the length of the effect and selecting the best drug regimen.
Administration, Oral↗
Evaluation of parathyroid autograft growth and function in hemodialysis patients.
The aim of our study was to evaluate the function and growth of parathyroid tissue autografted into the forearm of hemodialysis patients using several presently available methods. In a dynamic study, the secretory function of autografted tissue was evaluated in seven patients using either zero calcium dialysate or calcium infusion. In an additional prospective study, seven patients had repeated determinations of plasma immunoreactive parathyroid hormone (iPTH) concentration on samples from both forearms, a radionuclide evaluation of autograft function using thallium-201 chloride, and real time ultrasonography. Light microscopy analysis was performed in two patients. The dynamic study demonstrated that induction of hypocalcemia was followed by an increase, and induction of hypercalcemia by a decrease, in circulating iPTH in both forearms using three different radioimmunoassays, similar to what has been reported for normal parathyroid tissue. A significant gradient (ie, greater than 2.0) of plasma iPTH concentration in samples from both forearms was observed in only three out of the seven patients of the prospective study. Two of these patients disclosed an increased uptake of 201TI chloride at the site of autografted tissue and had an echographically detectable mass. In both, hyperplastic parathyroid tissue was removed. At present, the remaining third patient does not have other features of recurrent hyperparathyroidism. In conclusion, autotransplanted parathyroid tissue of hemodialysis patients shows an adequate response to physiologic stimuli such as hypo- and hypercalcemia. Dynamic tests, therefore, appear to be a useful tool in the assessment of its function. In addition, radionuclide and echographic studies may be reliable adjuncts in the detection of marked parathyroid autograft hyperplasia.
Adult↗