Biomedical subjects

J P Borel

Publications and source records attributed to J P Borel.

At least 19 recordsLinked to original sources

PubMed2000

A specific sequence of the noncollagenous domain of the alpha3(IV) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells.

The invasive properties of melanoma cells correlate with the expression of matrix metalloproteinases (MMPs) and their physiological modulators (tissue inhibitors of metalloproteinase and membrane-type MMPs) and with that of the alphaVbeta3 integrin. We investigated the effect of anterior lens capsule type IV collagen and of the alpha3(IV) collagen chain on the invasive properties of various tumor cell lines (HT-144 melanoma cells, HT-1080 fibrosarcoma cells). We demonstrated that anterior lens capsule type IV collagen or specifically the synthetic peptide alpha3(IV) 185-203 inhibited both the migration of melanoma or fibrosarcoma cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP and the beta3 integrin subunit.

Amino Acid Sequence↗

PubMed1999

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Amino Acid Sequence↗

PubMed1997

A cell binding domain from the alpha3 chain of type IV collagen inhibits proliferation of melanoma cells.

Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the alpha3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the alpha1, alpha2, alpha4, and alpha5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesion-promoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide alpha3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50-60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 microg/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the alpha3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS- (residues 189-191), which is unique to the alpha3 chain and may represent an important functional epitope.

Amino Acid Sequence↗

PubMed1997

All-trans-retinoic acid enhances collagen gene expression in irradiated and non-irradiated hairless mouse skin.

All-trans-retinoic acid (t-RA) can repair some of the tissue damage caused by chronic exposure of skin to UV radiation. In the present study, we have investigated its effect on collagen and collagenase gene expression in hairless mouse skin. Hairless mice (SKH-hr 1) were irradiated dorsally with increasing doses of UVB radiation (total, 4.8 J cm-2) for 10 weeks. The animals were then topically treated with 0.05% t-RA dissolved in a vehicle or with the vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatment. In our experimental conditions, UVB irradiation alone induced no changes in type I, III and VI collagen mRNA levels in dorsal and ventral skin. The mRNA level of collagenase I was also unchanged. Topically applied t-RA increased the steady state levels of type I and III collagen mRNA in irradiated and non-irradiated dorsal skin. The mean increase was about 2.2- and 2.7-fold in non-irradiated skin and 2.4- and 2.5-fold in irradiated skin for type I and III collagen mRNA respectively. The increase in irradiated skin was partly due to the vehicle alone, which exerted a stimulating effect on the steady state levels of alpha 1(I) and alpha 1(III) mRNA. The mRNA level of type VI collagen was also significantly increased by t-RA, but only in irradiated skin. The mRNA level of collagenase was significantly decreased only in irradiated t-RA-treated skin. In addition, t-RA exerted a systemic effect because the mRNA levels of collagen were enhanced by factors of 1.9 and 2.5 for alpha 1(I) and 2.0 and 2.0 for alpha 1(III) in the ventral skin of irradiated and non-irradiated animals respectively. This study leads to the conclusion that topical t-RA exerts directly and/or indirect effects on the expression of collagen genes in irradiated and non-irradiated hairless mouse skin.

Animals↗

PubMed1996

Modulation of protein synthesis by extracellular matrix: potential involvement of two nucleolar proteins, nucleolin and fibrillarin.

Fibroblasts cultivated in a collagen matrix exhibit a large decrease in the synthesis of most proteins, depending on transcriptional and posttranscriptional controls. We have previously shown that ribosomal RNA content and half-life were decreased in collagen lattice cultures. Here, we cultivated human dermal fibroblasts in monolayers and in lattices and studied by competitive RT-PCR analysis the expression of the nucleolar proteins nucleolin and fibrillarin, two key factors in ribosome processing and association. Nucleolin expression was found increased, and fibrillarin expression decreased, in collagen-lattice vs monolayer-cultured fibroblasts, with some variability according to the strains (+25 to +250% and -40 to -60%, respectively). These data suggest that a possible trouble of the association between neosynthesized rRNA and nucleolar proteins is, at least partly, responsible for the inhibition of protein synthesis induced by the extracellular matrix.

Cells, Cultured↗

PubMed1995

The binding of type I collagen to lymphocyte function-associated antigen (LFA) 1 integrin triggers the respiratory burst of human polymorphonuclear neutrophils. Role of calcium signaling and tyrosine phosphorylation of LFA 1.

Monoclonal antibodies to the alpha L beta 2 integrin inhibit the binding of type I collagen to PMN (polymorphonuclear neutrophil leukocytes) as well as the subsequent stimulation of superoxide production and enzyme secretion-elicited by this collagen. Pepsinized collagen still binds PMN but no longer stimulates them. The I domain of the alpha chain of the integrin is involved in the binding. Two sequences of the alpha 1(I) polypeptide chain of collagen participate in the process. Experiments of competitive inhibition by synthetic peptides showed that the sequence RGD (915-917) is used for binding to the cells and DGGRYY (1034-1039) serves to stimulate PMN. Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose-collagen confirmed the presence in PMN extracts of two proteins, 95 and 185 kDa, respectively, corresponding to the molecular weights of the beta 2 and alpha L chains of the integrin and recognized by their specific monoclonal antibodies. The transduction pathways depending on the alpha L beta 2 integrin do not involve a G protein (ruled out by the use of cholera and pertussis toxins), whereas the cytoskeleton was found to participate in the process, as evidenced by inhibition by cytochalasin B. After collagen stimulation, cytoplasmic inositol trisphosphate and calcium ion increased sharply for less than 2 min. The use of the inhibitors staurosporine and calphostin C demonstrated that protein kinase C was involved. Evaluation of the activity of this enzyme showed that, upon stimulation of PMN with collagen I, it was translocated to plasma membrane. Acrylamide gel electrophoresis of the protein bands corresponding to the integrin alpha L beta 2, followed by immunoblotting using monoclonal antibodies to phosphotyrosine, permitted us to demonstrate that, prior to stimulation by type I collagen, there was no phosphorylation, whereas after stimulation, both alpha L and beta 2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the beta 2 chain of the integrin, without stimulating O2-. production by these cells, whereas their stimulation by complete type I collagen induced the tyrosine phosphorylation of both alpha L and beta 2 subunits. The tyrosine phosphorylation of both integrin subunits during transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.

Amino Acid Sequence↗

PubMed1995

Glutamine increases collagen gene transcription in cultured human fibroblasts.

We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.

Amino Acids↗

PubMed1995

Protein synthesis in collagen lattice-cultured fibroblasts is controlled at the ribosomal level.

Fibroblasts cultivated in three-dimensional lattices exhibit a large decrease of protein synthesis, mainly through transcriptional control. However, no previous work was devoted to a potential ribosomal regulation. We evaluated ribosomal ribonucleic acid (RNA) in monolayer- and collagen lattice-cultured fibroblasts. After one week of culture, total RNA was 60% lower in lattice-cultured fibroblasts than in monolayer-cultured cells. The decrease was identical for 18 S and 28 S rRNA subfractions. The half-life of RNA was much shorter in collagen lattice-cultured fibroblasts than in monolayers. These results suggest that protein synthesis in lattice-cultured fibroblasts is partly regulated at the ribosomal level.

Adult↗

PubMed1995

Chronic UVB- and all-trans retinoic-acid-induced qualitative and quantitative changes in hairless mouse skin.

Histochemical and ultrastructural studies have already demonstrated that chronic exposure to UV radiation induces profound alterations in all structural elements of the skin and that topical all-trans retinoic acid (tRA) can substantially correct much of the tissue damage. However, previous biochemical studies on dermal components of the extracellular matrix have led to contradictory results, particularly with regard to the effect of chronic UV exposure. The aim of our study was to investigate changes in collagen content and other dermal modifications induced by tRA in irradiated and non-irradiated hairless mouse skin. Hairless mice were exposed to increasing doses of UVB for 10 weeks (the cumulative total dose was 4.6 J cm-2). After the UV irradiation period the animals were treated with 0.05% tRA or with ethanol-polyethylene glycol vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatments. The main clinical and histological changes induced by UVB exposure were erythema, wrinkling, keratosis and epidermal thickening. Following UVB exposure, tRA treatment did not improve the clinical aspect but increased the width of the dermal repair zone. Fibronectin, laminin and type I and VI collagens were detected by indirect immunofluorescence techniques in this zone. Type I and III collagens were quantitated in skin fragments after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Under our experimental conditions, UVB irradiation alone induced neither changes in total collagen nor in type I and III collagen levels. tRA treatment of irradiated skin significantly increased both type I and III collagen levels by factors of 1.33 and 1.88 respectively. The ratio of type III to types I + III increased significantly. Topical tRA also increased collagen type levels in non-irradiated hairless mouse skin. Type I collagen increased proportionally to type III. This study leads to the conclusion that topical tRA exerts direct or indirect effects on collagen metabolism in irradiated as well as non-irradiated hairless mouse skin.

Animals↗

PubMed1994

The alpha 3 chain of type IV collagen prevents activation of human polymorphonuclear leukocytes.

Our initial observation that type I collagen activates polymorphonuclear leukocytes (PMN) prompted the testing of the activating potential of type IV collagen. It was noted, however, that type IV collagen isolated from bovine lens capsule did not activate PMN but rather prevented their stimulation by N-formylmethionyl-leucyl-phenylalanine, phorbol myristate acetate, or type I collagen. This observation led to the present study, which demonstrates that the inhibitory effect of lens capsule type IV collagen resides in the noncollagenous (NC1) domain of the alpha 3 chain and specifically in the region comprising residues 185-203 of the NC1 domain of both the human and bovine molecules. Synthetic peptides from the same region of the NC1 domains of the alpha 1, alpha 2, alpha 4, and alpha 5 chains did not possess the inhibitory effect seen with the alpha 3 chain. The sequence S-N-S (residues 189-191) is unique to the peptide of the alpha 3 chain, and substitution of either serine with alanine abolishes the inhibition. Type IV collagen isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor, a molecule that lacks the alpha 3 chain, did not prevent PMN activation but instead stimulated the secretion of elastase and type IV collagenase. Incubation of PMN with intact lens capsule type IV collagen or a peptide comprising residues 185-203 of the alpha 3 (IV) chain resulted in a 3-fold increase of intracellular cAMP, whereas, Ca2+ levels remained unchanged. Incubating PMN with forskolin or with dibutyryl-cAMP resulted in the inhibition of O2- production and degranulation by PMN, thus mimicking the effects of type IV collagen and the alpha 3 (IV) 185-203 peptide. The data suggest that type IV collagen, through its alpha 3 chain, down-regulates PMN activation and thus decreases the potential for damage as these cells traverse the capillary wall. Our in vitro experiments suggest that the higher the content of the alpha 3 (IV) chain is in a basement membrane, the wider would be its capacity for self-protection.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells.

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.

Animals↗

PubMed1994

The effect of different collagens and of proteoglycan on the retraction of collagen lattice.

The effect of various collagens and proteoglycan on the formation and retraction of collagen lattices was tested. The most rapid aggregation of collagen molecules was observed by the use of the least cross-linked collagen fractions (ie pepsin-digested calf skin collagen type I). Lattices formed with more cross-linked collagens (acid soluble collagen-ASC, type III) contracted slowly and less intensively. Unpurified pepsinized cartilage extract containing collagen types II, IX and XI, some glycoproteins and proteoglycans formed lattices rather well. On the contrary, purified collagen type II as well as polymeric collagen (solubilized by denaturing conditions) did not form lattices at all. The lattice formation and retraction was intensified by addition of proteoglycan into the culture medium. The authors suggest that the kinetics of the lattice formation and retraction depends on the amount of collagen cross-links and the concentration of proteoglycan in the culture medium.

Collagen↗

PubMed1993

A fluorometric deoxyribonucleic acid assay for tridimensional lattice cultures of fibroblasts.

A fast and sensitive in situ assay of deoxyribonucleic acid in miniaturized lattice cultures of fibroblasts is described. Tridimensional collagen and fibrin lattices prepared in 24-well plates were seeded with 50,000 to 200,000 cells. Cultures were fixed with formaldehyde, rinced with isopropanol, and dried. DNA assay was performed directly in the wells by addition of 3,5-diaminobenzoic acid (DABA) reagent. A calibration curve was prepared with calf thymus DNA. Fluorescence of DNA-DABA was evaluated after 45 min incubation (excitation wavelength 420 nm, emission wavelength 490 nm). The method showed linear results from 0.5 to 10 micrograms DNA and proved sensitive for low cell numbers (50,000 per dish). DNA assay in monolayers and in different types of lattices showed that comparable results were obtained in the different models without interference of the extracellular matrix. This technique is regarded as a costless and efficient tool for evaluating the number of cells in lattices in basal conditions or under pharmacological stimulation.

Aminobenzoates↗

PubMed1993

Adhesion and activation of human neutrophils onto collagen chains separated by electrophoresis.

After separation of the various alpha chains of the collagens by SDS-PAGE, the binding of polymorphonuclear neutrophils (PMN) to these chains was detected by a double-antibody technique and the activation of PMN by nitro blue tetrazolium. All of the alpha chains tested were able to bind PMNs. The alpha 1 chain of type I collagen activated the PMN when it had not been treated with pepsin. Pepsinized types II and VI collagens did not activate PMN. The pepsinized alpha 1(III) chains and all three alpha chains from pepsinized type V collagen were able to activate PMN.

Cell Adhesion↗

PubMed1993

Adhesion and activation of human neutrophils on basement membrane molecules.

In a previous study, we found that type I collagen activates human polymorphonuclear neutrophils by binding to a membrane integrin [3]. The activation depends on two sequences, both contained in the alpha 1 (I) CB6 peptide, one is RGD, starting at residue 915, and the second is DGGRYY, starting at residue 1034 of the alpha 1(I) chain. We checked the effect of several other types of collagens, principally type IV collagen from several origins. The basement membrane from bovine lens as well as type IV collagen prepared from it by tartaric acid extraction did not activate the human neutrophils. In contrast, when neutrophils had been previously in contact with type IV collagen their activation by type I or the alpha 1(I) CB6 peptide, or the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine, was inhibited. This effect was abolished when type IV collagen had been previously treated by pepsin. On the other hand, the fractions of type IV collagen that resisted digestion by bacterial collagenase still exhibited this inhibiting effect. This effect probably explains the physiological property of neutrophils to cross vascular walls without being activated.

Amino Acid Sequence↗

PubMed1993

[Collagens: why such a structural complexity?].

The collagens are a family of extracellular fibrillar proteins, characterized by the presence of one or several domains termed "triple helix", that are made of three polypeptide chains folded around each other. They elicit a huge worldwide research activity, marked every year by the publishing of dozens of books and thousands of papers. This family is presently represented by more than 16 individualized types, all differing by their molecular structure and by the way helical and globular domains are arranged. In any case, however, at least one triple helical domain exists. It is formed by the association of three polypeptide chains, each of them containing a glycine every three residues and many proline or hydroxyproline residues, and attests for the belonging of the protein to the collagen group. These multiple molecular forms and their specific architecture raise questions that remain unsolved. Why is this triple helix structure adopted in the case of collagens? Is it because the simple alpha helix of protein cannot extend over more than a few nanometers and is not solid enough? Why not a double helix like that of DNA? It would probably not be rigid enough. Why are there many globular domains interspersed between fibrillar ones? Probably these domains are useful for the association of peptide chains in register prior to their folding, then they participate in the transport of the elementary molecules from the synthesizing cells to their final place in the connective tissue and, finally, they insert the molecules into their specific place inside the growing fibrils. Collagen fibres as they are evidenced by histological methods, for instance in tendons, are of complex structure. Most of their constituting sub-units are type I tropocollagen molecules but they also contain in their center a filament of type V collagen that seems to serve as a guide during their edification. On the surface of the fibres are molecules of type III collagen that limit the growth in diameter and also type XII molecules that serve to bind the fibres to the surrounding substances. The collagen type multiplicity is explained by their various functions (mechanical role for tendons and ligaments, functions of wrapping around muscle cells, basement membrane role as a support for endothelial cells, function of glomerular filter, etc.). The fact that every collagen type contains several different polypeptide chains remains poorly explained. It may serve for the orientation of every elementary molecule inside the complex array of the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Different regulation of collagen I gene transcription in three-dimensional lattice cultures.

Human skin fibroblasts were cultivated in confluent monolayers, retracting collagen lattices, retracting fibrin lattices and non-retracting fibrin lattices and the expression of messenger RNA specific for the alpha 1 chain of type I procollagen comparatively studied by Northern blot and dot blot hybridization. Two factors contribute to the lower level of procollagen messenger RNA in collagen lattices: the retraction and the nature of the fibrillar protein that constitutes the lattices. Fibrin lattices, when they do not retract, make as much collagen and procollagen mRNA as monolayer confluent cells.

Blotting, Northern↗

PubMed1992

Insulin-like growth factor-I (IGF-I) stimulates protein synthesis and collagen gene expression in monolayer and lattice cultures of fibroblasts.

Fibroblasts cultivated in three-dimensional tissue-like matrices are characterized by a slowed metabolism and a decrease of protein synthesis, unless they are submitted to physical tensions. We checked the effects of insulin like growth factor-I (IGF-I), known as a potent stimulator of mitogenesis and protein synthesis for many cell types, in various models of cultures: confluent monolayers, collagen lattices, non-retracting or retracting fibrin lattices. IGF-I (1-100 ng.ml-1) had no effect on cell divisions in lattice cultures. It was able to stimulate collagen lattice retraction when the medium was supplemented with low concentrations of serum. IGF-I at 10 or 100 ng.ml-1 stimulated collagen and non-collagen syntheses in all culture systems, but stimulation of collagen synthesis only began at the highest concentration (100 ng.ml-1) in retracted lattices. Northern blot and dot-blot analyses of mRNAs extracted from monolayer cultures of fibroblasts showed that IGF-I stimulated pro alpha 1(I) collagen synthesis at the pretranslational level. Cycloheximide (7.5 micrograms.ml-1) completely inhibited pro alpha 1(I) collagen gene expression induced by IGF-I. These results show that IGF-I is a potent stimulus for protein synthesis and collagen gene expression in monolayers and tridimensional cultures of fibroblasts, but that it exerts no mitogenic activity in tridimensional lattices. Synergistic associations of IGF-I with other growth factors will have to be found in order to reverse the quiescent status of fibroblasts in lattices.

Cell Count↗