Biomedical subjects

D Moscatelli

Publications and source records attributed to D Moscatelli.

At least 19 recordsLinked to original sources

PubMed2004

[Dental offices and environment quality].

AIM: The aim of this study was to survey, from a microbiological point of view, dental unit water, air and surface quality in public dental offices and in control environments in Milan. METHODS: We studied tap and dental unit water (from fountain, air-water syringe, turbine), at the beginning and at the end of monday and thursday morning activity; air quality with surface air system (SAS) in dental and control offices; handpieces holder, fountain block, arm of the light, dental trolley, inner and outer walls surfaces quality. RESULTS: Water from the dental unit waterline shows average exceeding the law limits 2-3 fold for total bacteriological counts at 37 degrees and 22 degrees, in all examined points, at the beginning of the working day. There is an improvement in the water contamination at the end of the activity. Pseudomonas aeruginosa is frequently found, total and fecal coliforms are absent, while Legionella pneumophila was found only in one control. Air and surfaces quality is quite good, especially in places with ventilation systems in function. CONCLUSION: Dental unit water is the most critical point among those monitored. Water quality has to be improved with specific projects.

Air Microbiology↗

PubMed2000

Fibroblast growth factor (FGF)-2 mediates cell attachment through interactions with two FGF receptor-1 isoforms and extracellular matrix or cell-associated heparan sulfate proteoglycans.

In the presence of FGF-2, cells in suspension expressing FGF receptor-1 will attach to monolayers of cells expressing heparan sulfates. This attachment provides physical evidence for the formation of a trimolecular complex between FGF-2, heparan sulfate, and FGF receptors. We have used this system to determine if receptor isoforms containing or lacking the first of three immunoglobulin-like domains are equally able to form complexes with FGF-2 and heparan sulfates. In the presence of FGF-2, cells expressing either isoform of the receptor were able to attach to monolayers of CHO cells expressing heparan sulfates. No attachment was observed in the absence of FGF-2 or if heparin was included in the incubation medium. Attachment of cells expressing the two receptor isoforms occurred at similar concentrations of FGF-2, and similar concentrations of heparin were required to disrupt the interactions. Thus, there appeared to be little difference between these receptor isoforms in their ability to form trimolecular complexes with FGF-2 and cell-associated heparan sulfates. We also found that, in the presence of FGF-2, cells expressing FGF receptor-1 are able to form complexes with both extracellular matrix and cell-surface heparan sulfates.

Animals↗

PubMed2000

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines.

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.

Animals↗

PubMed2000

Transforming growth factor-beta is an autocrine mitogen for a novel androgen-responsive murine prostatic smooth muscle cell line, PSMC1.

A prostatic smooth muscle cell line (PSMC1) was established from the dorsolateral prostate of p53 null mice. The cell line is nontumorigenic when inoculated subcutaneously, under the renal capsule or intraprostatically in syngeneic mice. These cells express alpha-smooth muscle actin (alpha-SMA), indicating their smooth muscle origin, and TGF-beta significantly enhances expression of alpha-SMA. The cells express both androgen receptor (AR) mRNA and protein, and respond mitogenically to physiological concentrations of androgens. PSMC1 cells produce significant amounts of TGF-beta, which stimulates growth by an autocrine mechanism. Dihydrotestosterone (DHT) increases proliferation of PSMC1 cells by promoting TGF-beta secretion. Considering the significant inhibitory effect of TGF-beta on prostatic epithelial cells and its stimulatory effect on the PSMC1 cells, we postulate that TGF-beta produced by prostatic smooth muscle cells may have a paracrine effect on the prostatic epithelium. We also postulate that TGF-beta may be involved in the etiology of benign prostatic hyperplasia (BPH) by stimulating excessive stromal proliferation. Line PSMC1 is the first reported androgen-responsive murine smooth muscle cell line. It will be useful for in vivo and in vitro experiments to study the mechanisms of androgen action on prostatic stroma and for delineating the interactions that occur between prostatic smooth muscle and epithelium that may lead to prostatic diseases such as BPH.

Androgens↗

PubMed1999

Inflammatory mediators regulate cathepsin S in macrophages and microglia: A role in attenuating heparan sulfate interactions.

BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases. The distribution of cathepsin S is restricted to cells from the mononuclear lineage both in the brain and in the periphery. Also, its protease activity is uniquely stable at neutral pH. MATERIALS AND METHODS: We compared the expression of cathepsin S, B, and L mRNAs in various undifferentiated and differentiated cells of mononuclear origin, and examined the modulation of these mRNAs by inflammatory mediators (lipopolysaccharide and various cytokines). In addition, the effect of these agents on cathepsin S protein levels and protease activity was also determined. Lastly, the ability of cathepsin S to process basement membrane components such as heparan sulfate proteoglycans in vitro and in vivo was assessed. RESULTS: Cathepsin S, B, and L mRNAs are expressed in mature macrophages and microglial cells and not in undifferentiated monocytes. Activators of macrophages negatively regulate all three transcripts. Consistent with this, treatment with these agents leads to a decrease in intracellular cathepsin S protein levels and activity. However, the same treatments result in stimulation of secreted cathepsin S activity. Cathepsin S is capable of degrading heparan sulfate proteoglycans in vitro. Also, when expressed in endothelial cells, cathepsin S autocrinely attenuates the basic fibroblast growth factor (bFGF)-mediated binding of FGF receptor containing cells to endothelial cells, by acting on basement membrane proteoglycans. CONCLUSIONS: Taken together, these data imply that cathepsin S is a regulatable cysteine protease that plays a role in the degradation of extracellular proteins, whose secretion from macrophages and microglia is increased by signals that lead to activation of these cells, and may be important in regulating extracellular matrix interactions. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p320.html

Animals↗

PubMed1996

Induction of urokinase-type plasminogen activator by fibroblast growth factor (FGF)-2 is dependent on expression of FGF receptors and does not require activation of phospholipase Cgamma1.

The roles of heparan sulfate proteoglycans and tyrosine kinase fibroblast growth factor (FGF) receptors in mediating the induction of plasminogen activator (PA) by FGF-2 were investigated using L6 myoblast cells that normally do not express detectable FGF receptors. PA was induced by FGF-2 in a dose-dependent manner in L6 cells expressing transfected FGF receptor-1 but not in nontransfected cells or cells transfected with the vector alone. The PA produced in these cells was characterized as urokinase-type PA (uPA). Thus, expression of a tyrosine kinase FGF receptor was required for induction of uPA. Internalization of FGF through heparan sulfates does not seem to be involved in this response as soluble heparin and suramin at concentrations which inhibited FGF-2 binding to heparan sulfates but not receptors did not affect the induction of uPA by FGF-2. Mutant receptors in which the tyrosine kinase was inactivated were not able to respond to FGF-2. In contrast, mutation of the site of phospholipase Cgamma1 (PLCgamma) binding in the receptor, which causes loss of PLCgamma activation, had no effect on uPA induction by FGF-2. These results suggest that PLCgamma activation is not required for induction of uPA by FGF-2.

Chlorates↗

PubMed1996

Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and heparan sulfates: downmodulation of FGF receptors by phorbol ester.

Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected a rapid loss of high affinity receptors. The ability to form bFGF-receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.

Animals↗

PubMed1995

Fibroblast growth factor-2 can mediate cell attachment by linking receptors and heparan sulfate proteoglycans on neighboring cells.

The myeloid 32D cell line, which grows in suspension and does not express FGF receptors or heparan sulfate proteoglycans, was transfected with the cDNA encoding FGF receptor-1 (32D-flg cells). When co-cultured with glutaraldehyde-fixed Chinese hamster ovary (CHO) cells, the 32D-flg cells remained in suspension in the absence of FGF-2 but attached to the CHO monolayer in the presence of 10 ng/ml FGF-2. In contrast, 32D cells transfected with the vector alone did not attach to the CHO monolayer in the presence of FGF-2. FGF-2-dependent attachment of 32D-flg cells was prevented by inclusion of 10 micrograms/ml heparin in the incubation medium and was diminished when CHO mutants in glycosaminoglycan synthesis or wild-type CHO cells treated with heparinase were used, indicating that the attachment occurred through FGF-2 interactions with heparan sulfates on the CHO cells. Attachment of 32D-flg cells to wild-type CHO cells was half-maximal at 0.4 ng/ml FGF-2 and was also observed with FGF-1 but not FGF-4. 32D-flg cells also attached to living CHO cells in a FGF-2-dependent manner, but attachment was transient at 37 degrees C. Induction of new proteins was not required for FGF-2-dependent attachment, since attachment occurred when the co-cultures were incubated at 4 degrees C and when the 32D-flg cells were preincubated with cycloheximide. FGF-2-dependent attachment of 32D-flg cells was also observed with Balb/C 3T3, NIH 3T3, and bovine capillary endothelial cells. We conclude that attachment is due to FGF-2 binding simultaneously to receptors on the 32D-flg cells and heparan sulfates on the CHO monolayers; thus, the FGF-2 acts as a bridge between receptor-expressing cells and heparan sulfate-bearing cells. In addition, induction of DNA synthesis in 32D-flg cells in response to FGF-2 was potentiated by the CHO-associated heparan sulfates to the same extent as by soluble heparin, indicating that this interaction has functional significance.

3T3 Cells↗

PubMed1995

Lipopolysaccharide inhibits activation of latent transforming growth factor-beta in bovine endothelial cells.

The activation of latent transforming growth factor-beta (TGF-beta) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-beta levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-beta activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 microM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-beta secreted into the culture medium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-beta expression as well as the suppression of surface activation of latent TGF-beta. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-beta did not occur in cells maintained in LPS-contaminated culture medium.

Animals↗

PubMed1994

Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding.

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin.

Animals↗

PubMed1994

Autocrine downregulation of fibroblast growth factor receptors in F9 teratocarcinoma cells.

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind 125I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of 125I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When 125I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000-160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells.

Animals↗

PubMed1994

Studies on FGF-2: nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin.

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1993

Expression of fibroblast growth factors and their receptors in acquired immunodeficiency syndrome-associated Kaposi sarcoma tissue and derived cells.

BACKGROUND: Fibroblast growth factors (FGF), such as basic FGF, have been implicated in the development of Kaposi sarcoma (KS) in vitro. The expression of several genes of the FGF family and their receptors in KS tumor lesions and KS-derived cells were evaluated. METHODS: Cultures of KS-derived cells were established. The expression of FGF family members and their receptors in these cells and in fresh biopsies from KS tumors was evaluated by reverse transcription polymerase chain reaction (RTPCR). The RTPCR products were confirmed by nucleotide sequencing. RESULTS: The expression of basic FGF and FGF receptor-1 (flg) was detected in all the samples tested. Acidic FGF (aFGF) and FGF-5 were detected in two of six and four of six KS tumor specimens, respectively, whereas both of these growth factors were expressed in all of the cell cultures, including six KS-derived cell cultures and human endothelial cells and smooth muscle cells. FGF-6 was expressed in two of six KS tumor specimens, but was not expressed in any of the cultured KS cells. Like flg, bek was expressed in all tissue samples and KS-derived cell cultures except in one KS specimen obtained from the patient's tongue showing expression of a high level of FGF-6. CONCLUSIONS: These results suggest that the expression of FGF in KS tumors with the coexpression of FGF receptors may provide a basis for autocrine and paracrine mechanisms contributing to the development of KS.

AIDS-Related Complex↗

PubMed1993

Involvement of the conserved acidic amino acid domain of FGF receptor 1 in ligand-receptor interaction.

The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flg delta A2 and flg delta A4, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flg delta A8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction.

Amino Acid Sequence↗

PubMed1993

Expression of int-2 oncogene in Kaposi's sarcoma lesions.

Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposi's sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Basic fibroblast growth factor (bFGF) dissociates rapidly from heparan sulfates but slowly from receptors. Implications for mechanisms of bFGF release from pericellular matrix.

The effect of heparin on the rate of binding of basic fibroblast growth factor (bFGF) to high affinity (receptor) and low affinity (heparan sulfate) binding sites on endothelial cells and CHO cells transfected with FGF receptor-1 or FGF receptor-2 was investigated. Radiolabeled bFGF bound rapidly to both high and low affinity sites on all three types of cells. Addition of 10 micrograms/ml heparin eliminated binding to low affinity sites and decreased the rate of binding to high affinity sites to about 30% of the rate observed in the absence of heparin. However, the same amount of 125I-bFGF bound to high affinity sites at equilibrium in the presence and absence of heparin. The effect of heparin on the initial rate of binding to high affinity sites was related to the log of the heparin concentration. Depletion of the cells of heparan sulfates by treatment with heparinase also decreased the initial rate of binding to high affinity receptors. These results suggest that cell-surface heparan sulfates facilitate the interaction of bFGF with its receptor by concentrating bFGF at the cell surface. Dissociation rates for receptor-bound and heparan sulfate-bound bFGF were also measured. Dissociation from low affinity sites was rapid, with a half-time of 6 min for endothelial cell heparan sulfates and 0.5 min for Chinese hamster ovary heparan sulfates. In contrast, dissociation from receptors was slow, with a half-time of 46 min for endothelial cell receptors, 2.5 h for FGF receptor-1, and 1.4 h for FGF receptor-2. These results suggest that degradative enzymes may not be needed to release bFGF from the heparan sulfates in instances where receptors and heparan sulfate-bound bFGF are in close proximity because dissociation from heparan sulfates occurs rapidly enough to allow bFGF to bind to unoccupied receptors by laws of mass action.

Animals↗

PubMed1992

Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms.

Basic fibroblast growth factor (bFGF) was internalized at a rapid rate by Chinese hamster ovary (CHO) cells that do not express significant numbers of high affinity receptors for bFGF as well as CHO cells that have been transfected with cDNA encoding FGF receptor-1 or FGF receptor-2. Internalization of bFGF was completely blocked by the addition of 10 micrograms/ml heparin in the parental CHO cells but only partially inhibited in cells expressing transfected FGF receptors. Bovine aortic endothelial cells also exhibit heparin-sensitive and heparin-resistant internalization of bFGF. The internalization of bFGF through the heparin-resistant pathway in CHO cells was efficiently competed by addition of unlabeled bFGF, was proportional to the number of receptors expressed, and approached saturation, suggesting that the heparin-resistant internalization was due to high affinity receptors. Internalization of bFGF through the heparin-sensitive pathway was not efficiently competed by unlabeled bFGF and did not approach saturation at concentrations of bFGF up to 50 ng/ml, properties similar to the interaction of bFGF with low affinity heparan sulfate binding sites on the cell surface. Internalization of bFGF in CHO cells not expressing FGF receptors was inhibited by heparin, heparan sulfate, and dermatan sulfate, the same glycosaminoglycans that block binding to cell-surface heparin sulfates. Internalization of bFGF in the parental CHO cells was inhibited at the same concentrations of heparin that block binding to cell-surface heparan sulfates. Finally, inhibition of the sulfation of CHO cell heparan sulfates by the addition of chlorate or digestion of CHO cell heparan sulfates with heparinase inhibited bFGF internalization in the parental CHO cells. These results demonstrate that bFGF can be internalized through a direct interaction with cell-surface heparan sulfates. Thus, there are two pathways for internalization of bFGF: high affinity receptor-mediated and heparan sulfate-mediated.

Animals↗

PubMed1992

Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

Amino Acid Sequence↗