©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Matrix Nonenzymatic Glycosylation Leads to Altered Cellular Phenotype and Intracellular Tyrosine Phosphorylation (*)

(Received for publication, October 3, 1994; and in revised form, November 10, 1994)

Goji Hasegawa Anne J. Hunter Aristidis S. Charonis (§)

From the Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota, 55455

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect of matrix nonenzymatic glycosylation on signal transduction and the cellular phenotype was examined. Human microvascular endothelial cells were plated on control or glycated basement membrane-like matrix. Cells exhibited a decrease in their ability to adhere and spread on modified matrix. The pattern of intracellular tyrosine phosphorylation was examined by Western Immunoblotting; a band with 65 kDa mobility exhibited a marked reduction of tyrosine phosphorylation in cells adherent to modified matrix. Immunoprecipitation experiments provided evidence that this band is paxillin, a member of focal adhesion proteins. Immunoprecipitation with antibodies against focal adhesion kinase (pp125), the enzyme that is thought to regulate paxillin tyrosine phosphorylation, also demonstrated a reduction in tyrosine phosphorylation of pp125. To confirm these biochemical data, adherent cells were examined for the distribution of paxillin, using immunofluorescence microscopy; paxillin was seen in focal points peripherally located in cells on normal matrix, but lacked this pattern in cells on modified matrix. Actin filaments were also disorganized in cells plated on modified matrix. These data suggest that matrix nonenzymatic glycosylation can interfere with and potentially alter cellular phenotype and intracellular signaling.


INTRODUCTION

In diabetes, hyperglycemia may affect many metabolic pathways, and each of these changes may have important consequences in the development of the various pathologic manifestations. A large body of evidence, including the results of the recent Diabetes Control and Complications Trial (DCCT), have documented that hyperglycemia and subsequent biochemical events correlate with the development of diabetic complications(1) . One of the mechanisms by which elevated sugar levels could compromise normal structure and function is the phenomenon of nonenzymatic glycosylation(2, 3) . Nonenzymatic glycosylation leads to the formation of primary (Amadori), intermediate and advanced glycosylation endproducts (AGEs)(^1)(4, 5) . These products are known to alter the structure and function of basement membrane macromolecules in vitro(6, 7) and are detected in situ in tissues from diabetic animals and humans(8, 9) .

It is well accepted that extracellular matrices and especially basement membranes are involved in interactions with cells and to a large extent determine the cellular phenotype(10, 11, 12) . During adhesion and spreading, cell receptors mainly of the integrin family interact with matrix macromolecules (13) and these interactions lead to important intracellular alterations. At the structural level, focal adhesions are forming and stress fibers are organized(14) . It has recently been shown that integrin-ligand interactions lead to tyrosine phosphorylation of focal adhesion kinase (pp125) (15, 16, 17, 18) , which in turn has the potential to phosphorylate other structural components of focal adhesions like paxillin and tensin(19, 20) . Changes in the cytoskeletal organization along with modifications of the activity of enzymes like pp125, may modify the activity of transcription factors and eventually may be crucial for the regulation of gene expression and cellular phenotype(21) . It is therefore of utmost importance to explore the possibility that hyperglycemia-induced matrix modifications could interfere with these processes and consequently result in changes of cellular functions.

Preliminary studies have provided indications that such changes may indeed occur(22, 23) . In this report, using an in vitro system of extracted basement membrane-like matrix, we examined the effects of matrix nonenzymatic glycosylation on the adhesion and spreading of human microvascular endothelial cells and we explored alterations in the signal transduction pathway, focusing on possible changes in intracellular phosphorylation patterns and the macromolecules involved in these modifications.


MATERIALS AND METHODS

Cell Culture

Human dermal microvascular endothelial (HME) cells were purchased from Cell Systems Corporation (Kirkland, WA). These cells were tested positive for low density lipoprotein receptor, factor VIII, and vimentin. Cells were maintained with the microvascular endothelial cell growth medium system available from the company, and all experiments were performed using cells between the 4th and 10th passage.

Preparation of Engelbreth-Holm-Swarm (EHS) Tumor Matrix Material and Nonenzymatic Glycosylation

EHS tumor grown subcutaneously in BALB/c mice (Jackson, Bar Harbor, ME) was removed and homogenized with a blender in 50 mM Tris, 3.4 M NaCl, 1 mM EDTA, 50 µg/ml PMSF, 50 µg/ml p-hydroxymercuribenzoic acid (all used as protease inhibitors), pH 7.4, at 4 °C. The homogenate was centrifuged at 3000 rpm for 30 min. The pellet was resuspended and washed for 5 more times with the same buffer. The pellet was resuspended in PBS containing 1 mM EDTA, 1 mMN-ethylmaleimide, 50 µg/ml PMSF, 50 µg/ml p-hydroxymercuribenzoic acid, pH 7.4, and stored at -80 °C before use.

The EHS matrix homogenate was dialyzed against PBS and then sonicated in an ice bath using a Sonifier 250 (Branson, Danbury, CT, USA). The sonicator was set at power output 7 with micro-tip and used for 10-s bursts with a 1-min cooling time. After total sonication time of 2 min, EHS matrix becomes small pieces up to 500 nm in diameter. This EHS matrix was incubated in 0.2 M phosphate buffer, containing 1 mM EDTA, 1 mMN-ethylmaleimide, 1 mM PMSF, and 0.02% sodium azide, pH 7.4, at 37 °C for various time intervals in the presence or absence of 1 MD-ribose. In some experiments, 1 M aminoguanidine (Sigma) was added to the 1 MD-ribose buffer; aminoguanidine is a nucleophilic hydrazine compound that inhibits the formation of AGEs(24) . In order to avoid oxidation-induced changes during the incubation period, all samples were degassed extensively and then bubbled with argon. D-Ribose has been used frequently because it is a reducing sugar much more reactive than glucose(25) . At the end of the incubation, samples were dialyzed against PBS at 4 °C. AGE contents of the samples were determined by measuring the fluorescence at 440 nm upon excitation at 370 nm (26) using Luminescence Spectrometer LS50B (Perkin Elmer) and standardized by protein contents of each sample.

Cell Adhesion and Spreading on EHS Matrix

96-Well plastic plates were coated with control and glycated EHS matrix by addition of 50 µl of 10 µg/ml matrix solution in PBS. The plates were left at 29 °C overnight to dry and were kept at 4 °C until used during the following 7 days. In preliminary experiments using radiolabeled EHS matrix, we have determined that under the experimental conditions used, the absence of sugar does not lead to differential extraction of matrix components and that glycation does not result in differential coating on plastic wells. Subconfluent human dermal microvascular endothelial cells were labeled with 500 mCi of [S]methionine (DuPont NEN) for 18 h. Then, the cultures of cells were pretreated with 25 µg/ml cycloheximide (Sigma) for 2 h to prevent synthesis and secretion of extracellular matrix components. Cells were washed with PBS and were detached by the addition of 0.05% trypsin, 0.75 mM EDTA solution. The detached cells were washed once in medium containing 10% calf serum, washed 3 times with serum free Dulbecco's modified Eagle's medium (Sigma) containing 2 mg/ml BSA fraction V (ICN Biomedicals Inc., Aurora, OH) and resuspended in this serum-free medium. Plates coated with the EHS matrix were incubated with PBS containing 2 mg/ml BSA, pH 7.4, for 2 h at 37 °C. Subsequently, 10,000 cells were applied on each well for 60 min. The nonadherent cells were aspirated at the end of the incubation, and the plates were washed 3 times with 200 µl of serum-free medium. Finally, the cells were lysed by incubating for 30 min at 60 °C with 100 µl/well of solution of 0.5 N NaOH, 1% SDS. The solution was then transferred in vials with scintillation fluid, and the radioactivity was counted in a scintillation counter. For cell spreading assays, cells were used without radioisotope labeling, and adherent cells were stained with Diff-Quik Stain Set (Baxter, Miami, FL). Cell perimeters were then measured by using Optomax camera connected to an Apple IIe computer.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting

For Western blotting, 10-cm culture dishes were employed and were coated with 10 ml of 10 µg/ml control or modified EHS matrix in the same procedure described above. Cells were plated at 5 times 10^6 cells/culture dish in serum-free Dulbecco's modified Eagle's medium, containing 2 mg/ml BSA. After 60-min incubation at 37 °C, nonadherent cells were aspirated and culture dishes were washed 3 times with PBS. The adherent cells were lysed with 300 µl of Laemmli sample buffer (80 mM Tris containing 3% SDS and 15% glycerol, pH 6.8). The samples were sonicated briefly to shear DNA. Samples containing equal protein concentrations were electrophoresed on 6 or 7.5% SDS-polyacrylamide gels under reducing condition by method of Laemmli(27) . After electrophoresis, gels were electrophoretically transferred to Immobilon-P membrane (Millipore, Bedford, MA) for 2 h at 0.2 A(28) . The membranes were blocked with 3% skim milk in PBS, pH 7.4, overnight at 4 °C and then incubated with monoclonal anti-phosphotyrosine antibody (UBI, Lake Placid, NY) at 1 µg/ml in blocking buffer for 2 h at room temperature. After washing with PBS, the membranes were incubated with 1:5000 dilution of sheep anti-mouse IgG conjugated with horseradish peroxidase (Sigma) in PBS containing 0.05% Tween-20 for 1 h at room temperature. The membranes were then washed with PBS containing 0.05% Tween-20, and the blots were developed using enhanced chemiluminescence (Amersham Corp.) according to the manufacturer's protocol.

Immunoprecipitation

Cells were lysed at 4 °C by scraping in 140 mM NaCl, 1 mM CaCl(2), 1 mM MgCl(2), 1 mM MnCl(2), 0.02% sodium azide, 50 mM Tris-HCl, pH 7.2 that contained 0.1 mM pervanade, 1 mM PMSF, 1 mMN-ethylmaleimide, 10 µg/ml leupeptin, and 1.0% Triton X-100. The lysates were clarified by centrifugation at 36,500 rpm for 1 h. To preclear the samples, 100 µl of protein A-agar (Sigma) conjugated with normal mouse IgG (Calbiochem, La Jolla, CA) was added to the samples and rotated for 3 h at 4 °C. The agar was sedimented by brief centrifugation at 12,000 times g, and the supernatant was transferred to the tube containing 10 µl of protein A-agar conjugated with monoclonal anti-paxillin antibody (Chemicon, Temecula, CA), and rotation continued for 2 h at 4 °C. The agar was pelleted by brief centrifugation at 12,000 times g and washed 4 times with wash buffer (400 mM NaCl, 1 mM CaCl(2), 1 mM MgCl(2), 1 mM MnCl(2), 0.02% sodium azide, 50 mM Tris-HCl, pH 7.2, that contained 0.1 mM pervanade, 1 mM PMSF, 1 mMN-ethylmaleimide, 10 µg/ml leupeptin, and 1.0% Triton X-100). Proteins were then released for SDS-polyacrylamide gel electrophoresis and Western blotting by boiling in Laemmli sample buffer for 5 min. To precipitate pp125, samples were precleared by protein A-agar conjugated with normal rabbit IgG (Sigma) and precipitated with polyclonal anti-pp125 (UBI, Lake Placid, NY) -conjugated protein A-agar. Released proteins in Laemmli sample buffer were applied for SDS-polyacrylamide gel electrophoresis and immunoblotted with monoclonal anti-phosphotyrosine antibody as described above. The immunoblots were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, 100 mM 2-mercaptoethanol at 55 °C for 30 min as recommended by the manufacturer, and then the blots were reprobed with anti-paxillin or anti-pp125 antibody.

Fluorescence Microscopy

Glass coverslips were coated with normal and glycated matrix, and cells (isolated as described above but without metabolic labeling) were allowed to interact for 60 min in serum-free medium. Unbound cells were gently washed off with PBS, and the remaining cells were fixed in 2% paraformaldehyde in Hanks' balanced salt solution for 30 min and then permeabilized for 2 min in 0.05% Triton X-100 and blocked for 30 min with 2% BSA in PBS. The coverslips were then incubated with anti-paxillin antibody (1:200) in PBS at room temperature. After 60 min, the coverslips were rinsed extensively in PBS and incubated with fluorescein-labeled phalloidin (1:200) (Sigma) and rhodamine-conjugated goat anti-mouse IgG (1:50) (Boehringer Manheim) in PBS for 30 min at room temperature. The coverslips were washed with PBS, rinsed in deionized water, and mounted on glass slides using 80% glycerol containing 50 mM Tris-HCl, pH 8.5, and 1 mg/ml phentlenediamine. The coverslips were examined with a Zeiss fluorescence microscope (Zeiss, Thornwood, NY) for detection of focal contacts; interference reflection microscopy was performed according to well established procedures(29) .

Statistics

All results are presented as means ± S.E. The Mann-Whitney U-test (30) was used to establish the statistical significance of difference in cell adhesion and spreading assays.


RESULTS

Accumulation of AGEs in the Matrix

Extracted EHS matrix was incubated for several time intervals in the absence of sugar or in the presence of either 1 M glucose or 1 M ribose. At the end of each time interval, the extent of formation of AGEs was monitored by increases in fluorescence, as described. A sharp and almost linear increase in fluorescence occurred during the first 5 days of incubation in ribose. At 5 days, the fluorescence intensity/µg of protein was 0.5 ± 0.1 in the control and 3.5 ± 0.2 in the sample incubated with ribose (n = 5). This increase was followed by a gradual but less dramatic increase for the next time periods, up to 20 days. Aminoguanidine, as expected, markedly reduced the accumulation of AGEs in the matrix incubated with 1 M ribose (0.8 ± 0.1 at 5 days, n = 5). Incubation in the presence of glucose resulted, as expected, in a more reduced accumulation of fluorescence, evident after 10 days of incubation; after 20 days of incubation, fluorescence reached the value of 2 days incubation in ribose (data not shown). From these data, and in order to keep the incubation time as short as possible, we considered as optimal conditions for most of the experiments the time interval of 5 days in 1 M ribose.

Adhesion and Spreading of HME Cells on Normal and Glycated Matrix

The ability of endothelial cells to adhere and spread on control or modified matrix was studied next. Matrix incubated for 0, 1, 2, 5, and 10 days in 1 MD-ribose was used for these assays. As shown in Fig. 1, cells adhered in a specific way on control matrix after a 60-min incubation in serum-free medium: 35% of added cells adhered, compared with only 4% of cells incubated on wells coated with BSA. The ability of cells to adhere was reduced in a fashion dependent on the extent of matrix incubation in 1 M ribose. For example, on matrix incubated for 5 days, only 20% of cells adhered specifically (Fig. 1). In order to evaluate cell spreading, perimeters of cells adherent to control matrix or matrix modified for different time intervals were traced. The results presented in Fig. 2demonstrate a dramatic decrease in cell spreading as a function of time of incubation in the reducing sugar. The findings described above demonstrate that matrix nonenzymatic glycosylation interferes with the initial events of cell-matrix interactions. These findings raised the possibility that alterations in intracellular signaling may follow.


Figure 1: Cell adhesion to glycated matrix. The ability of metabolically labeled human microvascular endothelial cells to adhere to EHS matrix incubated with 1 M ribose for various time period was measured. Results are expressed as percentage of adhering cells and shown as mean ± S.E. of five experiments, with a quadripulate determination for each value. The nonspecific adherence was 3.8 ± 0.4%, which was assessed by cell adherence to BSA-coated plastic plates. The values that do not share identical superscripts are significantly different. (p < 0.05-0.01). p < 0.01: 0 days versus 5 days.




Figure 2: Cell spreading on glycated matrix. Cell spreading was quantitated by measuring the perimeter of human microvascular endothelial cells allowed to spread for 60 min on EHS matrix incubated with 1 M ribose for various time periods. At least six randomly chosen fields were analyzed, and the perimeters of 30 cells on each condition were measured. This analysis was repeated three times with different plates. Results are shown as mean ± S.E., n = 30. Totally round cells exhibit a perimeter in the range of 70-80 µm. The values that do not share identical superscripts are significantly different. (p < 0.05-0.001). p < 0.001: 0 days versus 5 days.



Pattern of Tyrosine Phosphorylation on Glycated Matrix

In order to identify whether the morphological changes observed were coupled to changes in intracellular signaling, we studied the possible differences in tyrosine phosphorylation of intracellular proteins from cells on control versus modified matrix. As described in detail above, this approach included gel electrophoresis of cell extracts, electrobotting on Immobilon membranes, and screening with anti-phosphotyrosine antibodies.

In preliminary experiments, we studied the phosphotyrosine content of cell extracts as a function of incubation time. We observed that after 40-60 min, the amount of phosphotyrosine detected reached its peak (data not shown). Therefore, we decided to use the 60-min time interval in order to study the tyrosine phosphorylation profile.

In HME cells adhering to normal matrix, tyrosine phosphorylation was detected in bands with several mobilities; it was more pronounced in clusters of proteins between 115 and 130 kDa and at 65 kDa (Fig. 3, lane1). Cells adherent to matrix incubated for 5 days in 1 M ribose exhibited a similar pattern of tyrosine phosphorylation with one major difference; the extent of tyrosine phosphorylation on the 65 kDa band was markedly reduced (Fig. 3, lane2). Also, at 125 kDa, a relatively small reduction of the extent of tyrosine phosphorylation was apparent in cells on modified matrix. Nonadherent cells exhibited a different pattern, with much less tyrosine phosphorylation (Fig. 3, lane3).


Figure 3: Differences in tyrosine phosphorylation of intracellular proteins from human microvascular endothelial cells adhering to control versus glycated EHS matrix. Cells attached to normal (lane1), glycated matrix incubated for 5 days with 1 M ribose (lane2), or kept in suspension (lane3) for 60 min, were lysed and analyzed with Western immunoblotting as detailed in the text. In the cells on glycated matrix, the tyrosine phosphorylation of 65-kDa protein is markedly decreased; a slight decrease in tyrosine phoshorylation of 125-kDa protein was also observed. M, marker proteins; arrowheads indicate mobilities of 125 and 65 kDa.



Identification of Proteins Differentially Phosphorylated at Tyrosine Residues

The mobility of the major band that was differentially tyrosine phosphorylated was reminiscent of a macromolecule associated with focal adhesions, namely paxillin(31) . Since it is known that focal adhesions are important structures that provide a link between adhesion/spreading on extracellular matrices with cytoskeletal changes, we decided to test whether the 65 kDa band was indeed paxillin. To this purpose, we immunoprecipitated cell extracts using anti-paxillin antibodies and tested with gel electrophoresis followed by blotting and screening with anti-phosphotyrosine, as described above. The results are shown in Fig. 4A. Lane1 shows the material from cells adherent to control matrix, whereas lane2 shows extract from cells adherent to matrix modified by a 5-day incubation in ribose. It is clear that a major difference in paxillin tyrosine phosphorylation exists. The amount of paxillin present in each lane was monitored by stripping the blot from the anti-phosphotyrosine antibody and reprobing it with anti-paxillin antibody; the results shown in Fig. 4B demonstrate that the amount of paxillin present under the experimental conditions is comparable in all three permutations. Therefore the observed changes are primarily due to differential tyrosine phosphorylation.


Figure 4: Tyrosine phosphorylation of paxillin in human microvascular endothelial cells. Cells attached to normal (lane1) or glycated EHS matrix, incubated for 5 days with 1 M ribose (lane2), or kept in suspension (lane3) for 60 min, were lysed and immunoprecipitated with anti-paxillin. PanelA is immunoblotted with anti-phosphotyrosine. In panelB, the same blot was stripped and reprobed with anti-paxillin. Paxillin was tyrosine phosphorylated only in the cells on normal matrix. Ig, immunoglobulins.



To clarify the involvement of AGE formation in the matrix for the differential tyrosine phosphorylation of paxillin, the same experiment was performed using the matrix incubated with 1 M ribose in the presence of 1 M aminoguanidine. As shown in Fig. 5, tyrosine phosphorylation of paxillin was markedly restored in the cells adherent to matrix incubated in 1 M ribose when the inhibitor of AGE formation was present. This result demonstrates that AGE formation in the matrix plays the crucial role in the observed differential tyrosine phosphorylation of paxillin.


Figure 5: Restoration of tyrosine phosphorylation of paxillin in the cells attached to EHS matrix incubated for 5 days with 1 M ribose in the presence of 1 M aminoguanidine. Cells attached to the matrix for 60 min were lysed and immunoprecipitated with anti-paxillin. PanelA is immunoblotted with anti-phosphotyrosine. In panelB, the same blot was stripped and reprobed with anti-paxillin. Lane1, normal matrix; lane2, matrix incubated with 1 M ribose plus 1 M aminoguanidine; lane 3, matrix incubated with 1 M ribose alone. Ig; immunoglobulins.



Paxillin is a structural component of focal adhesions that is a putative substrate for the newly described enzyme, pp125. This molecule has a molecular mass of 125 kDa and is known to be tyrosine phosphorylated in response to cell adhesion to specific matrices. It is obvious from Fig. 3that a cluster of proteins with mobilities between 115 kDa and 130 kDa are tyrosine-phosphorylated in response to cell adhesion. This complex pattern may make detection of important changes rather difficult to evaluate. Because a minor difference in the extent of tyrosine phosphorylation was detected at 125 kDa (Fig. 3), we followed the same immunoprecipitation procedure using antibodies against pp125. The results shown in Fig. 6A indicate that pp125 is tyrosine phosphorylated in cells adherent to normal matrix (lane1), but the extent of tyrosine phosphorylation is decreased in cells adherent to glycosylated matrix (lane2). The amount of pp125 present in each lane was determined by reprobing the same blot with antibodies to pp125; as shown in Fig. 6B, no major differences in the amount of pp125 present in each lane were observed, suggesting differential phosphorylation of pp125.


Figure 6: Tyrosine phosphorylation of pp125 in human microvascular endothelial cells. Cells attached to normal (lane1) or glycated EHS matrix, incubated for 5 days with 1 M ribose (lane2), or kept in suspension (lane3) for 60 min, were lysed and immunoprecipitated with anti-pp125. PanelA is immunoblotted with anti-phosphotyrosine. In panelB, the same blot was stripped and reprobed with anti-pp125. Tyrosine phosphorylation of pp125 was detected only in the cells on normal matrix. Ig, immunoglobulins.



Paxillin Distribution and Cytoskeletal Organization in Cells Adherent to Normal or Glycated Matrix

In view of the altered tyrosine phosphorylation of paxillin, we seeked to determine whether it had any effect on its ability to cluster in focal adhesions and whether the actin cytoskeletal organization (stress fibers) was in any way modified. Fig. 7shows immunofluorescence staining for actin filaments (panelsA and C) and paxillin (panelsB and D), when cells were allowed to adhere on matrix unmodified (panelsA and B) or incubated for 5 days with 1 M ribose (panelsC and D). It can be observed that adherent cells on modified matrix exhibit much less organized actin filaments (Fig. 7C) compared with cells adherent on control matrix (Fig. 7A). Also, fine paxillin staining is localized to the end of actin filaments, indicating the beginning of formation of focal adhesions in cells plated on control matrix (Fig. 7B). Such pattern is almost undetectable in cells adherent on modified matrix (Fig. 7D).


Figure 7: Paxillin distribution and cytoskeletal organization in human microvascular endothelial cells adhering to normal or glycated EHS matrix, incubated for 5 days with 1 M ribose. Cells were plated for 60 min on normal (A and B) and glycated (C and D) matrix. Cells were stained for actin (A and C) and paxillin (B and D). Cells on glycated matrix exhibit much less organized actin filaments compared with cells attached to control matrix. Also, fine paxillin staining, localized to the end of actin filaments, is almost undetectable in cells adherent on glycated matrix. Bar equals 200 µm.




DISCUSSION

It is well established that nonenzymatic glycosylation alters the structural and the functional integrity of extracellular matrix(6, 7) . In the present report, we provide evidence that these matrix modifications may additionally affect cellular function and cause phenotypic changes. To our knowledge, this is the first report establishing that such modifications in the extracellular environment of a cell may lead to changes in the pattern of tyrosine phosphorylation.

The observed changes are likely to be mediated by the integrin family of adhesion receptors. There is increasing evidence that integrins do participate in information transfer and intracellular signaling(21, 32) . One of the processes in which integrin-mediated signaling has been implicated is the tyrosine phosphorylation of intracellular substrates. It has been reported that integrin clustering (generated either by antibodies or by adhesion to fibronectin or its cell binding fragments) can trigger tyrosine phosphorylation of an enzyme that is associated with focal adhesions, pp125(15, 16, 17, 18) . Furthermore, some structural components of focal adhesions are tyrosine phosphorylated after cell adhesion to extracellular matrices, and they are likely to be phosphorylated by focal adhesion kinase. These include paxillin, which is a major substrate for tyrosine kinases and tensin, which contains src homology 2 domains(19, 20) . Although the further downstream events of this signal transduction pathway are not clear, it is assumed that the differential phosphorylation induced by glycation of the extracellular matrix may affect gene expression. This can happen either as a consequence of cytoskeletal modifications or by directly or indirectly affecting transcriptional events in the nucleus. For example, it has been reported that integrin-mediated adhesion of monocytes to various matrices induces translocation of transcription factors(33, 34) . In addition to modification of the tyrosine phosphorylation pattern, it is possible that other changes in integrin-related signaling events might occur, such as cytoplasmic alkalization (35) or protein kinase C-mediated phosphorylation(36) .

In addition to the integrins, signaling pathways through AGE receptors may play an important role in the alteration of cellular phenotype. Endothelial cells are known to express such surface macromolecules that belong to the immunoglobulin superfamily(37) . It has been proposed that interaction of AGE receptors with their ligands in monocytes, induces production of cytokines like interleukin-1, insulin-like growth factor I, tumor necrosis factor alpha, in concentrations sufficient to alter cellular phenotype(38, 39) . It has recently been proposed that upon stimulation of these receptors by soluble AGE-modified BSA, activation of the NF-kappaB transcription factor follows(40) .

For our approach, we have used an in vitro system where matrix is heavily modified by using ribose, a reducing sugar far more reactive than glucose(25) . This allowed us to use much shorter time intervals for incubations and provided the ability to magnify putative changes, thus allowing a more conclusive analysis. Also, glucose-modified matrix inhibited the intracellular tyrosine phosphorylation. HME cells exhibited a reduction in paxillin tyrosine phosphorylation in the range of 10 and 50% of the control value when plated for 60 min on the EHS matrix incubated with 1 M glucose for 10 days and 20 days, respectively (data not shown). We believe that the alterations observed may occur even when the surrounding extracellular matrix is minimally modified because the change of phosphorylation observed correlates with the change of cell adhesion and spreading. In support of this notion are observations of reduction in adhesion and spreading of macrovascular endothelial cells adherent to minimally glycosylated laminin or type IV collagen(22) . However, the putative changes in intracellular signaling under such conditions remain to be examined.

Based on the data presented above, we would like to propose the hypothesis that changes in the extracellular matrix over a long time period may result in altered cellular phenotype; the type of modifications caused by such a mechanism may vary considerably, depending on the cell type affected. Among the possible changes one could envision differential expression of matrix components, matrix-degrading enzymes and/or their inhibitors, growth factors, cytokines, or enzymes dealing with oxygen raicals, like superoxide dismutase.


FOOTNOTES

*
This work was supported by Grants from the National Institutes of Health (DK-43569 and DK-36007), the Juvenile Diabetes Foundation International, and the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Laboratory Medicine and Pathology, University of Minnesota Medical School, Box 609 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455. Tel.: 612-625-0601; Fax: 612-626-3876.

(^1)
The abbreviations used are: AGE, advanced glycosylation endproduct; HME, human dermal microvascular endothelial cells; pp125, focal adhesion kinase; EHS, Engelbreth-Holm-Swarm; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Liz Wayner for advice and help with immunofluorescence and Dr Joji Iida for valuable advice.


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