(Received for publication, October 3, 1994; and in revised form, November 10, 1994)
From the
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.
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)()(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.
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.
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.
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.
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.
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.
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 , 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-
B 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.