Germana Dini

Proteoglycans in so-called cellulite.

ABSTRACT: Glycosaminoglycans are a group of polysaccharide chains covalently linked to proteins to form proteoglycan molecules with high water‐attracting properties. The ultra‐structural localization of glycosaminoglycans in the so‐called cellulite skin and in normal subjects was studied. Data show that there is increasing concentration of glycosaminoglycans in the cellulite skin, presumably leading to a rise in the amount of water retained in the skin in this disease.

Cell Invasion Is Affected by Differential Expression of the Urokinase Plasminogen Activator/Urokinase Plasminogen Activator Receptor System in Muscle Satellite Cells from Normal and Dystrophic Patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti–u-PA and anti–u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA–induced invasion, as well as in u-PA–induced proliferation.

Involvement of glycosaminoglycans in detachment of early myeloid precusors from bone-marrow stromal cells

Fibroblast-like cells were obtained by in vitro cultivation of needle aspirations of human bone-marrow. These cells show a unique composition of coat-associated glycosaminoglycans: 10% chondroitin 4-sulfate, 30% hyaluronic acid and 60% heparan sulfate which were resolved and characterized by electrophoresis, nitrous acid treatment and enzymatic degradation. Chondroitin 4-sulfate is the only glycosaminoglycan detectable on the surface of mature granulocytes, whereas the immature cells do not seem to possess surface glycosaminoglycans. Immature hemopoietic cells can adhere on to marrow-derived fibroblast cells, whereas mature granulocytes cannot. Treatment with mucopolysaccharidases of both mature leukocytes and marrow stromal cells can interfere in these adhesive relationships, suggesting a role of glycosaminoglycans in regulating short-range interactions during hematopoiesis.

The Mr 17 500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.

Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential.

We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF‐2) in the expression of transformation‐related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF‐2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF‐2 or the four HMW FGF‐2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage‐independent growth and increased invasive potential. However, HMW FGF‐2‐expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF‐2 forms or LMW FGF‐2 alone. We have grown the different cell lines under a selective pressure of N‐(phosphonacetyl)‐l‐aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl‐P‐synthetase/aspartate transcarbamylase/dihydro‐orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF‐2 and/or HMW FGF‐2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear‐targeted HMW FGF‐2 has a pivotal role in such a cooperation. J. Cell. Physiol. 193: 64–72, 2002.

Synoviocytes from osteoarthritis and rheumatoid arthritis produce plasminogen activators and plasminogen activator inhibitor-1 and display u-PA receptors on their surface

The production of plasminogen activators and their inhibitors was studied in vitro in osteoarthritic (OA) and rheumatoid arthritic (RA) synovial fibroblasts (SF), obtained from RA and OA patients undergoing joint surgery. Subcultured SF were cultivated for 2, 4, 6, 8, 10 and 13 days and the medium assayed for the presence of both plasminogen activators (PAs) and plasminogen activator inhibitor-1 (PAI-1). The presence of urokinase-Plasminogen Activator (u-PA) receptors (u-PAR) on the surface of synovial cells was investigated by radio-ligand binding assay and cross-linking and by transmission electron microscopy (TEM) of a gold-u-PA complex. Our results showed a low production of tissue-type-Plasminogen Activator (t-PA) in both OA and RA SF, but relatively high levels of u-PA, until confluence, both in OA and in RA. SF were also able to produce plasminogen activator inhibitor in large amounts, in particular in RA since the very beginning of the culture. Receptors for u-PA were evident on both RA and OA SF. Our data show that SF in vitro produce mainly u-PA, the most important plasminogen activator involved in tissue modifications. The demonstration of u-PA receptors on the surface of OA and RA SF represents a step forward in the understanding of the possible role of fibrinolytic and tissue destructive proteinase cascade in joint inflammation.

Selective localization of receptors for urokinase amino-terminal fragment at substratum contact sites of an in vitro-established line of human epidermal cells

We have shown the presence of surface receptors for the amino-terminal fragment (ATF) of human urokinase-type plasminogen activator (u-PA) on an in vitro-established cell line of human epidermal origin by both radio-binding assays with human 125I-u-PA-ATF and transmission electron microscopy of a gold-u-PA complex. On the basis of cross-linking experiments with 125I-u-PA-ATF and subsequent autoradiography of the gels we have observed that such receptors are not spontaneously released into the culture medium. The treatment with phosphatidylinositol-specific phospholipase C induces the release of the receptor, which behaves as a glycosyl phosphatidyl inositol(GPI)-anchored protein. Phase-partitioning experiments on cell lysates have shown that the receptor partitions into the detergent phase. By detaching cell monolayers with the chelating agent EDTA we have prepared the cell-substratum contact sites of these cells, which represent only the 3.5% of the surface membrane of monolayered cells. Such plasma membrane remnants are highly selected since they contain about 43% of total u-PA-ATF binding sites. Such binding sites show the same biochemical and morphological characteristics of u-PA-ATF receptors observed in the monolayered cells, thus indicating that u-PA is selectively concentrated at the level of cell-substratum contacts. This is likely to enable directional proteolysis for cell migration and invasion.

Interaction of urokinase with specific receptors abolishes the time of commitment to terminal differentiation of murine erythroleukaemia (Friend) cells.

We have shown the presence of specific receptors for human urokinase on the surface of mouse erythroleukaemic cells. The receptor number increases when undifferentiated cells undergo hexamethylene‐bisacetamide induced differentiation, while affinity between receptor and ligand does not change. A monoclonal antibody against the 17 500 mol wt fragment of the non‐catalytic chain of urokinase (A chain) inhibits the specific binding, as we have previously shown in A431 cells. We have found that, upon the simultaneous addition of both urokinase or the A chain and hexamethylenebisacetamide, commitment is initiated immediately without the lag shown by control cultures of undiffer‐entiated cells treated with the low molecular weight inducer alone. A series of mechanisms possibly involved in the action of urokinase on mouse erythroleukaemic differentiation are also discussed.

Modulation of surface-associated urokinase: Binding, interiorization, delivery to lysosomes, and degradation in human keratinocytes

Receptor-mediated endocytosis of urokinase-type plasminogen activator (u-PA) was characterized with the human keratinocyte cell line NCTC, by both biochemical and ultrastructural methods. Binding to specific cell surface receptors at low temperature occurs with both catalytically active and inhibited u-PA. At 37 degrees C a single cohort of bound u-PA molecules is rapidly reduced at the surface level by both membrane dissociation and intracellular accumulation of the ligand, with no difference between active and inhibited u-PA. After a short lag period, both intact u-PA and u-PA degradation products are released into the culture medium. In the continued presence of native and inhibited u-PA at 37 degrees C the cumulative ligand uptake largely exceeds the total cellular capacity of binding sites measured at low temperature, consistent with receptor recycling. Catalytically inhibited u-PA shows a reduced interiorization rate, consistent with a requirement of an intact catalytic site which becomes evident in the presence of multiple cycles of endo-exocytosis. In the presence of a molar excess of anti-plasminogen activator inhibitor-type 1 (PAI-1) antibodies the interiorization rate is similar to that observed with catalytically inhibited u-PA, suggesting that PAI-1 molecules can modulate the intracellular accumulation of u-PA in this cell line. Parallel electron microscopy studies of a u-PA-colloidal gold complex have shown that membrane-associated u-PA molecules are concentrated in clusters before invagination of the underlying membrane to form endosomes which then fuse with lysosomes, where at least a part of u-PA degradation is likely to occur. Also, ultrastructural studies have confirmed the decrease in intracellular u-PA accumulation after inhibition of u-PA catalytic site. We conclude that cell surface-associated u-PA modulation in human keratinocytes involves ligand binding, uptake, and degradation, mediated by the classic receptor system for u-PA A chain, which can be modulated by membrane-associated PAI-1 molecules.

Mitochondrial alterations induced by selenium in guinea pig myocardium

Abstract The ip administration of selenium in guinea pigs produced a selective myocardial mitochondria damage. The damage was prevented by ip administration of methionine and pyruvate. These observations could be useful in the treatment of selenium poisoning.