scholarly journals Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling.

1980 ◽  
Vol 85 (3) ◽  
pp. 839-852 ◽  
Author(s):  
A Gonzalez-Noriega ◽  
J H Grubb ◽  
V Talkad ◽  
W S Sly
Keyword(s):  
Cell Surface ◽  
Lysosomal Enzyme ◽  
Human Fibroblasts ◽  
Acid Hydrolases ◽  
Surface Binding ◽  
Exogenous Enzyme ◽  
Chloroquine Treatment ◽  
Enhanced Secretion ◽  
Normal Human ◽  
The One

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta-glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.

scholarly journals Secretion of β-N-acetylglucosaminidase isoenzymes by normal human fibroblasts

1978 ◽  
Vol 173 (2) ◽  
pp. 433-439 ◽  
Author(s):  
P Willcox
Keyword(s):  
Lysosomal Enzyme ◽  
Human Fibroblast ◽  
Lysosomal Enzymes ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
The Other ◽  
Normal Human Fibroblast ◽  
Acid Hydrolases ◽  
Human Fibroblast Cultures ◽  
Normal Human

1. Secretion of the lysosomal enzyme beta-N-acetylglucosaminidase (EC 3.2.1.30) by normal human fibroblast cultures was linear with respect to time up to 96h. 2. Two forms of the A isoenzyme of beta-N-acetylglucosaminidase were found in the culture medium. One form was similar to the isoenzyme found in other extracellular fluids, such as plasma and tears, the other resembled the intracellular (lysosomal) enzyme. The presence of the two isoenzymes in the culture medium appears to reflect two distinct secretory processes. 3. It is suggested that plasma acid hydrolases may be destined for incorporation into lysosomes in a manner analogous to that described for the packaging of lysosomal enzymes by fibroblasts.

scholarly journals A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

1991 ◽  
Vol 11 (5) ◽  
pp. 2547-2557 ◽  
Author(s):  
P W Cook ◽  
P A Mattox ◽  
W W Keeble ◽  
M R Pittelkow ◽  
G D Plowman ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Human Keratinocytes ◽  
Mitogenic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Surface Binding ◽  
Mammary Tumor Cell ◽  
Human Keratinocyte ◽  
Heparin Sulfate ◽  
Normal Human ◽  
Autocrine Factor

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts

1988 ◽  
Vol 8 (10) ◽  
pp. 4234-4242
Author(s):  
B L Allen-Hoffmann ◽  
C L Crankshaw ◽  
D F Mosher
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Cell Surface ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Tgf Beta ◽  
Surface Binding ◽  
Amino Terminal ◽  
Cell Surface Binding

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.

scholarly journals Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

1988 ◽  
Vol 8 (10) ◽  
pp. 4234-4242 ◽  
Author(s):  
B L Allen-Hoffmann ◽  
C L Crankshaw ◽  
D F Mosher
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Cell Surface ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Tgf Beta ◽  
Surface Binding ◽  
Amino Terminal ◽  
Cell Surface Binding

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.

scholarly journals Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface

1998 ◽  
Vol 334 (2) ◽  
pp. 345-353 ◽  
Author(s):  
Kaisa LEHTI ◽  
Jouko LOHI ◽  
Heli VALTANEN ◽  
Jorma KESKI-OJA
Keyword(s):  
Cell Surface ◽  
Matrix Metalloproteinase ◽  
Human Fibroblasts ◽  
Proteolytic Processing ◽  
Gelatinase A ◽  
Surface Binding ◽  
Fibrosarcoma Cells ◽  
Subsequent Activation ◽  
Membrane Type

Human fibroblasts and HT-1080 fibrosarcoma cells express membrane-type-1 matrix metalloproteinase (MT1-MMP), the cell surface activator of gelatinase A, in separate forms of 63 kDa, 60 kDa and in some cases 43 kDa. In the present work the interrelationships between MT1-MMP processing and gelatinase A activation were analysed using HT-1080 fibrosarcoma cells as a model. It was found that MT1-MMP was synthesized as a 63 kDa protein, which was constitutively processed to a 60 kDa active enzyme with N-terminal Tyr112, as shown by immunoprecipitation, immunoblotting and sequence analyses. Co-immunoprecipitation results indicated that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhibited by calcium ionophores. Although the active MT1-MMP was required for cell surface binding and activation of gelatinase A, it was inefficient in activating gelatinase A in fibroblasts or in control HT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT1-MMP were found to be critical for gelatinase A activation. In HT-1080 cells, MT1-MMP was further processed to an inactive, 43 kDa cell surface form when overexpressed, or when the cells were treated with PMA. Under these conditions, the activated gelatinase A was detected in the culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activation and degradation/inactivation) of MT1-MMP and MT1-MMP/TIMP-2 relationships at the cell surface are important regulatory levels in the control of gelatinolytic activity.

scholarly journals Proteolytic Processing of the β-Subunit of the Lysosomal Enzyme, β-Hexosaminidase, in Normal Human Fibroblasts

1989 ◽  
Vol 264 (6) ◽  
pp. 3380-3384
Author(s):  
D V K Quon ◽  
R L Proia ◽  
A V Fowler ◽  
J Bleibaum ◽  
E F Neufeld
Keyword(s):  
Lysosomal Enzyme ◽  
Human Fibroblasts ◽  
Proteolytic Processing ◽  
Β Subunit ◽  
Normal Human

scholarly journals Identification of cell surface dipeptidylpeptidase IV in human fibroblasts

1983 ◽  
Vol 216 (1) ◽  
pp. 177-183 ◽  
Author(s):  
M Saison ◽  
J Verlinden ◽  
F Van Leuven ◽  
J J Cassiman ◽  
H Van den Berghe
Keyword(s):  
Cell Surface ◽  
Human Fibroblasts ◽  
Gel Filtration ◽  
Living Cells ◽  
Hydrophobic Domain ◽  
Dipeptidylpeptidase Iv ◽  
Pig Kidney ◽  
Crossed Immunoelectrophoresis ◽  
Kidney Enzyme ◽  
Normal Human

An antigen with dipeptidylpeptidase IV activity was identified at the surface of normal human fibroblasts. Hydrophobic interaction electrophoresis in phenyl-Sepharose revealed that the enzyme contained a hydrophobic domain, while lactoperoxidase-catalysed iodination with 125I of living cells indicated that the protein was located at the cell surface. Crossed immunoelectrophoresis with specific antibodies of acid-extracted or papain-treated cells showed a shift of the dipeptidylpeptidase IV peak to a faster mobility. The molecular properties of the fibroblast enzyme were clearly different from those described for dipeptidylpeptidase IV from other tissues and species. Fibroblast dipeptidylpeptidase IV contained two different disulphide-linked subunits, of apparent Mr values 125000 and 135000 (denatured and reduced). In gel filtration, an Mr of about 400000 was observed for the unreduced molecule. The enzymic properties of fibroblast dipeptidylpeptidase IV were very similar to those of the well-characterized pig kidney enzyme. Activity towards glycyl-L-prolyl-beta-naphthylamide was inhibited 50% by 0.023 mM-di-isopropylphosphorofluoridate. L-Alanyl-L-alanyl-beta-naphthylamide was hydrolysed ten times more slowly than glycyl-L-prolyl-beta-naphthylamide.

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