Biomedical subjects

A Wasteson

Publications and source records attributed to A Wasteson.

At least 19 recordsLinked to original sources

PubMed2001

Interleukin-6 enhances transforming growth factor-alpha mRNA expression in macrophage-like human monocytoid (U-937-1) cells.

We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-alpha) and that the steady-state levels of TGF-alpha mRNA as well as TGF-alpha protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF-alpha expression in keratinocytes. In the present study we investigated whether TGF-alpha expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation. U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF-alpha mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF-alpha gene was accompanied by release of TGF-alpha protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF-alpha as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF-alpha gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF-alpha protein release or pro-TGF-alpha surface expression. We conclude that since IL-6 causes increased steady-state levels of TGF-alpha mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA.

Cell Differentiation↗

PubMed2000

Both intra- and extracellular Ca2+ participate in the regulation of the lateral diffusion of the PDGF-beta2 receptor.

When the receptors for platelet-derived growth factor (PDGF) are activated they aggregate, become tyrosine-phosphorylated and elicit a cascade of down-stream signals, including mobilization of Ca2+ from intra- and extracellular stores. Receptor mobility in the plane of the membrane is a prerequisite for receptor aggregation and further signalling. Using human foreskin fibroblasts (AG 1523) and fluorescence recovery after photobleaching (FRAP), we therefore assessed the lateral mobility characteristics of PDGF-beta2 receptors by their diffusion coefficient (D), and fraction of mobile receptors (R). This was done on cells stimulated with either normal human serum (NHS) or PDGF under different Ca2+-conditions. The results suggest that both intra- and extracellular free Ca2+ influence the mobility characteristics of the PDGF-beta2 receptor. Interestingly, the extracellular Ca2+ seems to impose general restrictions on the mobility of receptors, since R increased when extracellular Ca2+ was quenched with EGTA, whereas intracellular clamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affected D. When both intra- and extracellular Ca2+ were quenced, D remained low and R high, further supporting the proposition that they achieve distinct effects. Inhibition of tyrosine phosphorylation with Erbstatin, partly inhibited the NHS effects and released PDGF-induced receptor immobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGF induced changes in intracellular free [Ca2+]. In view of the present data it might have important effects on the state of the receptor in the membrane, for instance by regulating its lateral mobility, communication with other receptors and signalling functions in the membrane.

Animals↗

PubMed1999

Suramin blocks growth-stimulatory effects of platelet-derived growth factor on malignant fibrous histiocytomas in vitro.

The pattern of susceptibility of malignant cells to the cytostatic drug suramin is not fully clarified. Therefore, in the present paper we have assessed the effects of suramin on the growth of eight cell lines derived from human malignant fibrous histiocytomas, by measuring DNA synthesis. The effect of suramin (10-200 microg/ml) on cells either unstimulated, or stimulated with platelet-derived growth factor (PDGF)-AA (10 ng/ml), PDGF-BB (10 ng/ml) or 10% fetal calf serum was studied. Four out of five cell lines unable to thrive without external growth factors showed growth inhibition by suramin. The two cell lines able to grow under serum-free conditions were unaffected by high-dose suramin. The exposure to suramin, at 200 microg/ml, abolished the growth stimulation caused by PDGF-AA and -BB. In contrast, a low dose of suramin (50 microg/ml), with or without PDGF, caused growth-stimulating effects in some cell lines. Our results indicate that high doses of suramin inhibit growth of malignant fibrous histiocytomas in vitro and that suramin exerts its growth-inhibitory effects on cells dependent on external growth factors. Low-dose treatment with suramin, however, may instead promote growth in both serum-dependent and -independent tumor cell lines.

Antineoplastic Agents↗

PubMed1999

CR3, FcgammaRIIA and FcgammaRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca(2+), phospholipase D and tyrosine phosphorylation.

Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcgammaRIIA and FcgammaRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcgammaRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcgammaRIIA Pansorbins induced an even weaker signal. However, FcgammaRIIA induced strong phosphorylation of p72(syk) whereas FcgammaRIIIB induced only a very weak p72(syk) phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72(syk) was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72(syk) that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72(syk) phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcgammaRIIA and FcgammaRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72(syk) is an early signal in the cascade induced by FcgammaRIIA but not by CR3.

Antibodies, Monoclonal↗

PubMed1999

Effects of human platelet-derived growth factor-AB on sarcoma growth in vitro and in vivo.

Platelet-derived growth factor (PDGF) has been proposed to play an important role in the growth of tumors. In order to study the effects of PDGF-AB on tumor growth in vivo, sarcoma-bearing mice were treated with PDGF-AB. The tumors, a malignant fibrous histiocytoma and an osteosarcoma, had functional PDGF receptors in vitro, as demonstrated by stimulation of PDGF-AB using a [3H]thymidine incorporation assay. Immunohistochemistry also revealed that both sarcoma xenografts expressed PDGF receptors. The tumor-bearing mice were given human PDGF-AB for 14 days, either continuously by an intraperitoneally placed mini-osmotic pump, or by daily injections. No effects on tumor growth in vivo were observed, as measured by tumor volume, autoradiography or cell cycle distribution. The histological appearance and ploidy of the tumors remained unaltered. The results indicate that, although the tumor cells are stimulated by PDGF-AB in vitro, the in vivo milieu or tumor growth pattern may render the tumors less susceptible to exogenously administered PDGF-AB in vivo.

Aged↗

PubMed1998

Uptake of 125I-PDGF-AB to the blood after extravascular administration in mice.

The kinetics of exogenously given 125I-platelet-derived growth factor-AB (PDGF-AB) was studied in mice. 125I-PDGF-AB was injected either intraperitoneally, intramuscularly or subcutaneously and the resulting concentrations of 125I-radioactivity monitored in the blood at different times. The serum levels of 125I-radioactivity rose to a maximum 2-4 hours after injection, before decreasing. Precipitation of serum with trichloroacetic acid demonstrated that 50 per cent or more of the 125I remained in macromolecular form. Further, gel chromatography studies showed that the molecular size of the labelled material in serum, three hours after injection, was the same as that of the original 125I-PDGF-AB. In addition, a low-molecular weight fraction was observed, indicating the presence of degradation products. The largest proportion of degraded material was obtained after subcutaneous administration. The absence of partially degraded 125I-labelled fragments, e.g. 125I oligopeptides, indicates complete rather than limited degradation and suggests that the 125I-PDGF-AB had been processed by cellular uptake. It is concluded that extra-vascularly given PDGF-AB in mice is taken up into the blood in intact macromolecular form. This finding suggests that it is possible to administer PDGF extravascularly to obtain a prolonged increase in the concentration of intact PDGF in the blood.

Animals↗

PubMed1998

Human malignant fibrous histiocytomas in vitro: growth characteristics and their association with expression of mRNA for platelet-derived growth factor, transforming growth factor-alpha and their receptors.

Eight human malignant fibrous histiocytomas were examined in vitro, in order to relate their growth properties to mRNA expression for platelet-derived growth factor (PDGF), PDGF receptor (PDGF-R), transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R). Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that all cell lines expressed mRNA for PDGF-R alpha and/or PDGF-R beta; six cell lines expressed mRNA for the PDGF-A chain, with one cell line coexpressing PDGF-B chain mRNA; seven cell lines expressed mRNA for TGF-alpha whereas six cell lines expressed EGF-R mRNA. Conditioned medium from three cell lines contained PDGF; none of the cell lines released TGF-alpha. Two cell lines grew without serum requirements; whereas both expressed mRNA for PDGF, PDGF-R, TGF-alpha and EGF-R, other cell lines, unable to grow without serum, showed the same combination of growth factor/growth factor receptor expression. The two cell lines able to grow without serum were also shown to be stimulated by the addition of PDGF-BB. These findings show that simultaneous expression of mRNA for a growth factor and its receptor does not necessarily imply an autocrine or paracrine loop. However, two of our cell lines fulfil the requirements of possible PDGF-related autocrine and paracrine regulation.

Adult↗

PubMed1996

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts.

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D +/- standard error of the mean, SEM) of 4.2 +/- 0.2 x 10(-10) cm2/s, and 1.8 +/- 0.2 x 10(-10) cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3 +/- 0.3 x 10(-10) cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1 +/- 0.8 x 10(-10) cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.

Antioxidants↗

PubMed1995

Transforming growth factor-alpha (TGF-alpha) in human bone marrow: demonstration of TGF-alpha in erythroblasts and eosinophilic precursor cells and of epidermal growth factor receptors in blastlike cells of myelomonocytic origin.

The expression of transforming growth factor-alpha (TGF-alpha) in human differentiating leukemic cell lines and in circulating human eosinophils prompted the search for an analogous function in normal human bone marrow (BM) cells. Immunohistochemistry, using a monoclonal antibody directed to the mature form of the TGF-alpha molecule, showed TGF-alpha on the erythroblasts of normal donors. This novel property of erythroid cells was found on cells at all stages of maturation, most clearly on nucleated forms but to some extent also on erythrocytes within the BM. The presence of membrane-bound TGF-alpha on erythroblasts was confirmed by immunomagnetic cell sorting with polyclonal TGF-alpha antibodies; the recovered cells consisted almost entirely of erythroblasts. Using another monoclonal antibody directed to TGF-alpha, immunohistochemistry showed a different pattern of positive cells including eosinophilic precursor cells, in accordance with earlier findings in blood eosinophils. In addition, the TGF-alpha immunoreactivity was shown in promyelocytes and neutrophilic myelocytes. The presence of epidermal growth factor (EGF) receptor mRNA in BM cells was demonstrated by reverse transcription polymerase chain reaction, whereas EGF receptor-carrying cells were recognized by immunohistochemistry, using polyclonal antibodies directed to the cytoplasmic part of the EGF receptor. The EGF receptor-positive cell constituted about 3% of the nucleated BM cell population. It was classified as a blastlike cell of myelomonocytic origin by morphologic criteria and CD68 positivity. Our results may indicate a novel function of TGF-alpha in erythrocytic differentiation.

Antibodies, Monoclonal↗

PubMed1995

Growth factor release during preparation and storage of platelet concentrates.

The platelet content of platelet-derived growth factor (PDGF), a mitogen stored in the alpha-granules, was studied during preparation and storage of platelet concentrates (PC) and compared to the growth-promoting activity of platelets, beta-thromboglobulin (beta-TG) and lactate dehydrogenase (LD). We compared PC prepared from platelet-rich plasma (PRP-PC; n = 10) and from buffy coat. Two different pre-preparation storage periods of the buffy coat were used: 4 h (BC-PC:4h; n = 10) and 24 h (BC-PC:24h; n = 5). The platelet content of PDGF and beta-TG was measured by a RIA technique and the growth-promoting activity by incorporation of 3H-thymidine in stimulated fibroblasts. The platelet content of PDGF, beta-TG and the growth-promoting activity of the platelets decreased in a similar way during preparation and storage of PRP-PC (31 +/- 2, 35 +/- 2 and 33 +/- 7%, respectively, at day 5 of storage; mean +/- SEM). The release of LD was minor (3.9 +/- 0.5% at day 5). At day 1 of storage the platelet content of PDGF was significantly better preserved in BC-PC:4h than in BD-PC:24h (88 +/- 2 and 81 +/- 3%, respectively; p = 0.03). Comparing BC-PC:4h and PRP-PC we found a significantly better preservation of PDGF in BC-PC:4h until day 3 of storage (80 +/- 2 and 75 +/- 1%, respectively at day 3; p = 0.046). In conclusion the preparation of PC according to the PRP method initially induces a higher loss of PDGF, and hence of the growth-promoting activity, than the BC method.

Blood Platelets↗

PubMed1995

Production of transforming growth factor alpha by human leukemia cells (HL-60 and U-937) during monocytic differentiation.

We have previously demonstrated that human promyelocytic HL-60 cells express transforming growth factor-alpha (TGF-alpha) during granulocytic differentiation. The present experiments were carried out in order to determine whether cells differentiated towards monocytes/macrophages will analogously express the TGF-alpha proto-oncogene product. HL-60 cells were induced to differentiate with 1 microM 1,alpha 25-dihydroxycholecalciferol (vitamin D3), and the human monocytoid cell line, U-937, was induced with 1 microM retinoic acid (RA), 0.1 microM vitamin D3, or 0.16 microM phorbol-12-myristate-13-acetate (PMA), ie experimental protocols known to induce monocyte/macrophage differentiation in these cells. In HL-60 cells, lacking constitutive TGF-alpha mRNA, vitamin D3 caused expression of the TGF-alpha gene and protein as demonstrated by Northern blot analysis and enzyme-linked immunoabsorbant assay (ELISA). In U-937 cells, showing constitutive TGF-alpha expression, RA but not vitamin D3 or PMA, caused marked increase in TGF-alpha mRNA (approximately 5-fold) and protein (approximately 3-fold) levels. In both cell lines the increase in TGF-alpha mRNA was evident within 24 h and continued throughout the observation period. Thus, it is established that differentiation of human leukemia cells towards monocytes/macrophages may be accompanied by TGF-alpha gene and protein expression in vitro. This is in conformity with the observed ability of mature activated macrophages to produce TGF-alpha.

Antigens, CD↗

PubMed1994

Transforming growth factor alpha expression in normal human blood eosinophils: differential regulation by granulocyte-macrophage colony-stimulating factor and interleukin-3.

We have previously demonstrated a constitutive expression of transforming growth factor alpha (TGF-alpha) in normal human blood eosinophils, both at the mRNA and protein level. This may indicate a novel function of the eosinophil, the regulation of which has not been clarified. Therefore human white blood cells (WBC) were treated with potential regulators of eosinophil function. Northern blot analysis demonstrated that human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) caused a time and dose-dependent 2- to 3-fold increase of TGF-alpha mRNA levels, in relation to incubation in the absence of cytokine; maximal response was attained within 4 h of incubation. In contrast, IL-5 failed to influence the expression of the TGF-alpha gene. In situ analysis of GM-CSF- or IL-3-stimulated cells showed that eosinophils remained the sole cell type expressing TGF-alpha mRNA. However, whereas GM-CSF significantly induced, within 1 h, release of immunoreactive TGF-alpha protein, IL-3 was insufficient in this respect. In conclusion, our findings indicate that expression of TGF-alpha gene and protein in normal blood eosinophils is differently regulated by GM-CSF and IL-3.

Dose-Response Relationship, Drug↗

PubMed1993

Expression of the transforming growth factor alpha protooncogene in differentiating human promyelocytic leukemia (HL-60) cells.

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

Cell Differentiation↗

PubMed1993

Production of transforming growth factor alpha by normal human blood eosinophils.

Transforming growth factor alpha (TGF-alpha) is a pleiotropic factor mediating numerous cellular responses in normal and transformed cells. This includes differentiation, proliferation, migration, and formation of extracellular matrix. TGF-alpha has been demonstrated in circulating eosinophils from the idiopathic hypereosinophilic syndrome and in differentiating promyelocytic leukemia cells in vitro. Whether TGF-alpha production also occurs in normal human blood cells is not known. Northern blot analysis showed that normal human white blood cells consistently expressed the TGF-alpha gene in 47 out of 47 donors. Cell preparations enriched in mononucleated cells, and devoid of granulocytes, showed no TGF-alpha mRNA. In situ hybridization experiments assigned the TGF-alpha gene expression to the eosinophils; 100% of the eosinophils and no other cell types were specifically recognized by the complementary human TGF-alpha riboprobe. White blood cells, incubated at 37 degrees C for up to 6 hours, released immunoreactive TGF-alpha to the incubation medium, as determined by ELISA. In contrast, no TGF-alpha protein was detected in the incubation medium of mononuclear cells. It is concluded that TGF-alpha is constitutively produced and released by normal human blood eosinophils. TGF-alpha provided by eosinophils, may participate in the inflammatory reaction by interacting with mesenchymal and epithelial cells, thus promoting fibrosis or neovascularization.

Eosinophils↗

PubMed1991

Suramin inhibits growth of human osteosarcoma xenografts in nude mice.

The effect of suramin on tumor growth and morphology in two different human osteosarcoma xenografts (L-I OSM and L-II OSM) grown in BALB/cA-nu/nu mice was studied. Suramin (total dose, 720 mg/kg) given by i.p. injection (60 mg/kg/dose) for up to 9 weeks significantly inhibited osteosarcoma cell growth in both tumors, suramin-treated tumors showing only one-third or less of the volume of nontreated controls. Cell cycle distribution of tumor cells measured by DNA flow cytometry demonstrated that suramin treated caused accumulation of cells in the S and G2 phases of the cell cycle, in both L-I OSM and L-II OSM. In the aneuploid L-II OSM tumor suramin preferentially inhibited the growth of aneuploid cells, leading to a decrease in the ratio of aneuploid to diploid cells. Both osteosarcomas retained their histological appearance and the liver, spleen, heart, and kidneys of the treated animals were unaffected by suramin. These results are compatible with the view that suramin inhibits the growth of human osteosarcomas by cytostatic effects.

Animals↗

PubMed1991

Binding and biological effects of insulin, insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities.

Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of 125I-insulin was 0.9 +/- 0.2% of total 125I-insulin added, and the IC50-value was estimated to 8.9 pmol/l. The insulin analogue B10 Asp tended to be more potent than insulin in displacing 125I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of 125I-insulin-like growth factor-I after 4 h incubation at 10 degrees C was five times higher than the specific binding of insulin (4.4 +/- 0.4% of total 125I-insulin-like growth factor-I added), and the IC50-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing 125I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for 3H-thymidine incorporation into DNA: insulin-like growth factor-I greater than B10 Asp greater than insulin-like growth factor-II greater than insulin greater than or equal to B28 Asp greater than B9 Asp/B27 Glu greater than proinsulin. The maximal effect of insulin-like growth factor-I on 3H-thymidine incorporation was 71 +/- 16% higher than the maximal effect of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Lateral diffusion of PDGF beta-receptors in human fibroblasts.

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding "normal" and "starved" cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10(-10) cm2s-1 and greater than 90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

Cell Line↗

PubMed1990

The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.

Reconstruction of cartilage with perichondrium depends on the chondrogenic property of the perichondrial fibrocytes. The present investigation concerns the conditions for the differentiation of fibrocytes into chondrocytes both in vivo and in vitro. For the in vivo studies specimens of rib and auricular perichondrium from adult rabbits were wrapped round silicon rods which were enclosed in dialysis bags. One was placed in the suprapatellar pouch of the knee joint and one was placed intraperitoneally in each rabbit. After two months the bags were extracted, the perichondrium prepared for microscopic examination, and the chondrogenesis evaluated. In vitro the perichondrium was divided into small pieces and incubated with tissue culture medium. The medium was supplemented with fetal calf serum, together with epidermal growth factor, platelet derived growth factor, synovial fluid, or with human serum albumin (control group). After three weeks the explants were prepared for microscopy. Chondrogenesis was judged by the degree of cellular enlargement, capsule formation, deposition of matrix, and activation of the outer fibrocytic layer. In vivo, good cartilage development was found in all specimens placed in the knee joint but, in those placed intraperitoneally, little if any chondrogenesis was seen. In vitro profound differentiation occurred in all cultures supplemented with epidermal growth factor and platelet derived growth factor. An equivalent differentiation was found in perichondrium that had been incubated with synovial fluid. We conclude that the differentiation of perichondrial fibrocytes is initiated in vitro by growth factors. In addition, we have shown that synovial fluid contains factors that promote and enhance the development of cartilage from perichondrium.

Animals↗