1 Departments of Medicine and 2 Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, and Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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Glomerular
hypertension and glomerular hypertrophy act early and synergistically
to promote glomerular injury in diabetes. We have previously shown that
increased renal kinin production contributes to the glomerular
hemodynamic abnormalities associated with diabetes. Glomerulosclerosis,
characterized by mesangial cell proliferation and matrix expansion, is
the final pathway leading to renal failure. The signal(s) initiating
mesangial cell proliferation is ill defined. In the present study, we
utilized immunofluorescence, immunoprecipitation, and immunoblotting
techniques to identify substrates that are tyrosine phosphorylated in
response to bradykinin action in mesangial cells. Immunofluorescence
microscopy of mesangial cells stained with anti-phosphotyrosine
(anti-PY) antibodies following bradykinin treatment
(109-10
6
M) revealed a dose-dependent increase in the labeling of cytoplasmic and nuclear proteins. Immunoprecipitation with anti-PY, followed by
immunoblot revealed bradykinin-induced tyrosyl phosphorylation of
tubulin and mitogen-activated protein kinase (MAPK). Confocal microscopy of mesangial cells stained for MAPK indicated that bradykinin stimulation resulted in translocation of MAPK from the
cytoplasm to the nucleus by 2 h. These data demonstrate that bradykinin
action results in the tyrosine phosphorylation of cellular proteins in
mesangial cells and suggest a role for tubulin and MAPK in the
signaling cascade of bradykinin leading to altered mesangial function.
tyrosine phosphorylation; immunocytochemistry; B2-kinin receptor
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INTRODUCTION |
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DIABETIC GLOMERULOSCLEROSIS, characterized by mesangial cell proliferation and extracellular matrix expansion, is thought to be the final pathway leading to end-stage renal failure (5, 23). This destructive pathway leads to focal and eventually diffuse glomerulosclerosis, independent of the initial pathogenic mechanism (5). Since hyperperfusion and hyperfiltration are associated with some of the conditions that predispose to development of these glomerular abnormalities, it was postulated that hemodynamic factors are responsible for the initiation and progression of glomerular injury (6). A number of recent observations suggest that glomerular hypertrophy is closely linked to the development of glomerulosclerosis (18, 39). Although glomerular hypertrophy frequently accompanies glomerular hypertension, some evidence suggests that mesangial cell expansion is the predominant factor in the development of glomerulosclerosis (11, 29, 43). Although glomerular hypertrophy and glomerular hypertension may exert independent effects, recent evidence indicates that they may also act synergistically to cause glomerular injury and accelerated sclerosis (44).
The initiating and sustaining signals that may link increased glomerular pressure and mesangial cell hypertrophy are not fully understood. With regard to the hemodynamic changes that may initiate renal injury, we have accumulated considerable evidence that supports a role for the renal kallikrein-kinin system as a mediator of hemodynamic changes occurring in diabetes. Diabetic rats with moderate hyperglycemia show increased renal and urinary excretion of active kallikrein and kinins, in conjunction with reduced renal vascular resistance and increased glomerular filtration rate (GFR) and renal plasma flow (RPF) (15). Acute treatment of these hyperfiltering diabetic rats with aprotinin, a kallikrein inhibitor, or with a B2-kinin receptor antagonist, reduced GFR and RPF and increased renal vascular resistance (15, 19). Moreover, type 1 diabetic patients with hyperfiltration show increased excretion of active kallikrein compared with diabetic patients with normal GFR (14).
A role for kinins as growth promoting factors in mesangial cell proliferation was only recently explored. Although bradykinin was shown to directly stimulate DNA synthesis and cell number in mesangial cells, the signaling mechanisms leading to mesangial cell proliferation in response to bradykinin are still undefined (9). The B2-kinin receptor is a member of the seven transmembrane G protein-coupled receptor superfamily that lacks the tyrosine kinase domain (31). Since protein tyrosine phosphorylation is one of the primary signaling events utilized by growth factors to initiate cellular responses, we initiated studies to identify substrates that are tyrosine phosphorylated in response to bradykinin in mesangial cells.
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METHODS |
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Rat glomerular mesangial cells were prepared by a modification of the
method of Lovett et al. (27). Kidneys from male Sprague-Dawley rats
(75-100 g body wt) were removed under sterile conditions, and
cortices were isolated, minced finely, and passed through consecutive
sterilized stainless-steel sieves with pore sizes of 180, 150, 90, and
75 µm. Glomeruli collected on top of the 75-µm sieve are 95%
pure, as assessed by light microscopy. The glomeruli were
then incubated in Hanks' balanced salt solution plus
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 7.4, containing 0.3% collagenase at 37°C for 2 h
to remove epithelial cells, leaving the glomerular cores containing
mesangial and endothelial cells. The cores were diluted in 1.5 ml RPMI
1640 medium per kidney, containing 0.6 mU/ml insulin, 100 U/ml
penicillin, 100 µg/ml streptomycin, 0.25 mg/ml amphotericin, and 20%
fetal calf serum (FCS), conditions which favor growth of mesangial
cells. Cells were incubated at 37°C in a humidified atmosphere of
95% air-5% CO2. Mesangial cells
were identified by the following criteria: they stained positive for
actin, myosin, and desmin and negative for cytokeratin and factor VIII
antigens. Mesangial cells isolated by this procedure were homogeneous
and were used in all studies between passages 2-4.
Immunocytochemistry. Mesangial cells
grown to subconfluence on sterile microscope cover slips were rendered
quiescent by growing them for 2 days in serum-free media. The cells
were then stimulated with bradykinin at different concentrations
(109-10
6
M, n = 6) for various time intervals
(0-30 min, n = 6). Following treatment, the cells were washed with cold phosphate-buffered saline
(PBS) and fixed with 4% paraformaldehyde plus 0.1% glutaraldehyde for
10 min at room temperature and permeabilized using serial dilutions of
ethanol. Nonspecific binding was blocked by incubating cells for 10 min
in PBS containing 1% bovine serum albumin (BSA). In studies examining
the effects of bradykinin on tyrosyl phosphorylation, mesangial cells
were incubated overnight at 4°C with a 1:50 dilution of primary
anti-rabbit polyclonal anti- phosphotyrosine (anti-PY) antibody (33),
followed by washing with PBS containing 1% BSA and incubation at room
temperature for 1 h with a rhodamine-conjugated goat anti-rabbit
immunoglobulin G (IgG) secondary antibody diluted 1:50 (Chemicon
International). Cells were washed three times with cold PBS, 10 min
each, and mounted with Fluorsave (Calbiochem, San Diego, CA) on glass
slides. The stained cells were viewed under epifluorescence microscopy.
Confocal microscopy. In studies examining the effects of bradykinin on mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinases (ERK) nuclear translocation, mesangial cells were incubated overnight at 4°C with 1:50 dilution of primary monoclonal anti-Pan ERK antibody (Transduction Laboratories, Lexington, KY), followed by incubation at room temperature for 1 h with a rhodamine-conjugated goat anti-mouse IgG secondary antibody diluted 1:50. The cells were washed, mounted onto slides using Fluorsave, and viewed by laser-scanning confocal microscopy on a Zeiss Axioskope (MRC 1000, Bio-Rad) using a ×63 objective.
[32P]phosphate
incorporation and immunoprecipitation.
Quiescent mesangial cells grown to subconfluence in 60-mm culture
dishes were incubated with
[32P]orthophosphate (1 mCi/60-mm dish) for 3 h at 37°C. After the incubation period, cells
were washed with phosphate-free buffer and stimulated with bradykinin
(107 M,
n = 3) for 5 min. The cells were then
washed with cold PBS and immediately frozen in liquid nitrogen. The
frozen cell monolayer was scraped and solubilized in 400 µl of
solubilization buffer [50 mM HEPES, pH 7.4, 10 mM sodium
pyrophosphate, 100 mM NaF, 4 mM sodium orthovanadate, 1% (vol/vol)
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 U/ml
aprotinin], incubated for 15 min at 4°C and centrifuged at
3,000 revolutions/min (rpm) for 10 min. The supernatant was harvested
as the cytosolic fraction that was subjected to immunoprecipitation.
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RESULTS |
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Bradykinin stimulates tyrosyl phosphorylation of
mesangial cells: immunocytochemical studies. Protein
phosphorylation reactions play a pivotal role in amplifying and
disseminating incoming signals throughout the cell. To examine the
pattern of tyrosyl phosphorylation in response to bradykinin, quiescent
mesangial cells grown on coverslips were stimulated with bradykinin
(106 M) for various times
(0-30 min). The cells were fixed, permeabilized, and incubated
overnight at 4°C with 1:50 dilution of primary anti-PY antibody,
followed by incubation at room temperature for 1 h with a
rhodamine-conjugated secondary antibody diluted 1:50. Cells were viewed
under epifluorescence microscopy with a ×60 objective. Bradykinin
produced a rapid and intense tyrosyl phosphorylation of cytoplasmic and
nuclear proteins within 1 min of stimulation, decreasing in intensity
over 30 min (Fig. 1). Tyrosine-phosphorylated filaments spanning the entire cytoplasmic domain were
observed in the bradykinin-stimulated cells. Bradykinin produced a
dose-dependent increase in tyrosyl phosphorylation with a significant
immunofluorescence occurring with
10
9 M and near maximal
response at 10
7 M (Fig.
2, B-E). No fluorescent staining was
detected using secondary antibodies in the absence of primary
antibodies (Fig. 2, F).
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To identify the tyrosine-phosphorylated filaments observed in response
to bradykinin, double immunolabeling experiments were carried out.
Since microtubules are mainly composed of tubulin polymers, mesangial
cells treated with bradykinin
(106 M) for various times
were stained with polyclonal anti-PY and monoclonal anti-tubulin
primary antibodies. Primary antibodies were then detected with
rhodamine-conjugated goat anti-rabbit (PY) and fluorescein-conjugated
goat anti-mouse (tubulin) secondaries. As shown in Fig.
3, bradykinin stimulated the tyrosine
phosphorylation of filamentous structures. The same cytoskeletal
elements stained with anti-PY were also stained with anti-tubulin
antibodies, indicating microtubules are tyrosyl phosphorylated in
response to bradykinin stimulation.
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Immunoprecipitation and immunoblotting
studies. To further characterize the cytoplasmic and
nuclear proteins that were tyrosyl phosphorylated in response to
bradykinin, quiescent mesangial cells grown in 10-cm dishes were
stimulated with bradykinin
(108 M) for 5 min. Cell
lysates (500 µg protein) were preincubated for 5 min with Pansorbin,
followed by an overnight immunoprecipitation with 5 µg of anti-PY
antibodies at 4°C. The immunocomplex was resolved by
SDS-PAGE, and the separated proteins were transferred to PVDF
membranes and immunoblotted with recombinant anti-PY RC 20 H
antibodies. The labeled proteins were visualized using the ECL reagent.
Addition of bradykinin to mesangial cells resulted in a rapid increase
in tyrosine phosphorylation of several proteins (Fig.
4).
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To identify whether the 56- and 42-kDa protein bands that were tyrosine
phosphorylated in response to bradykinin were tubulin and MAPK,
respectively, mesangial cells were labeled with
[32P]orthophosphate
and were stimulated with bradykinin
(107 M) for 5 min.
Cytoplasmic and membrane fractions were prepared as described above and
subjected to selective immunoprecipitation with anti-MAPK antibodies
and anti-tubulin antibodies, respectively. Bradykinin stimulated the
phosphorylation of both the 42- and 44-kDa forms of MAPK in the
cytoplasmic fraction and tubulin in the membrane fraction, indicating
that both MAPK and tubulin may serve as substrates for
bradykinin-stimulated tyrosine phosphorylation in mesangial cells (Fig.
5, A and
B).
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Activation and nuclear translocation of MAPK by
bradykinin. The MAPK signal transduction pathway
represents an important mechanism by which growth factors regulate cell
growth. To examine whether bradykinin stimulates the tyrosyl
phosphorylation of MAPK, mesangial cells were treated with bradykinin
(108 M) for 5 min. Proteins
from the cell lysate were separated on SDS-PAGE and transferred to PVDF
membranes and immunoblotted with anti-phospho-MAPK antibodies (New
England Biolabs). Bradykinin resulted in tyrosyl phosphorylation and,
hence, activation of MAPK (Fig. 6).
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The nuclear consequences of ERK translocation include activation of the
transcription factor Elk-1, which results in production of c-fos
leading to activation of the AP-1 complex (37, 12). For this to occur, ERK must translocate to the nucleus. We utilized immunocytochemistry and confocal microscopy to examine whether bradykinin induces nuclear translocation of ERK. Figure
7, A and B, represents an optical section
through the nucleus of mesangial cells treated with vehicle or
bradykinin (107 M) for 2 h,
respectively. In unstimulated cells, ERK localization was entirely
cytoplasmic and concentrated in the perinuclear region of the cells.
Treatment of mesangial cells with bradykinin resulted in the nuclear
translocation of MAPK, based on the intense immunofluorescent staining
within the nucleus (Fig. 7B). No
fluorescence was detected in mesangial cells treated with bradykinin in
the absence of primary antibody (Fig.
7C).
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DISCUSSION |
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Growth factor receptor activation initiates biochemical cascades or signal transduction pathways that ultimately lead to changes in gene expression and induction of the mitogenic response. Identification of the signaling pathways is crucial to understanding the regulatory mechanisms involved in cell growth. Protein phosphorylation and dephosphorylation appears to be a primary signaling mechanism utilized by many growth factor receptor families that are linked to protein tyrosine kinases and those that are coupled to G proteins (34, 42). In the present study, we demonstrate that bradykinin stimulates the tyrosyl phosphorylation of a number of proteins in mesangial cells. Immunocytochemical and biochemical evidence revealed that microtubules are tyrosyl phosphorylated on tubulin in response to bradykinin. Bradykinin also induces the activation and nuclear translocation of MAPK in mesangial cells. These findings provide the initial evidence that bradykinin stimulates early growth events in mesangial cells.
The mechanism(s) through which bradykinin stimulates tyrosyl phosphorylation is as yet undefined. The B2-kinin receptor is a member of the seven transmembrane G protein-coupled receptor superfamily that lacks the tyrosine kinase homology region (31). Upon binding to its receptors in mesangial cells, bradykinin activates phospholipase C via a heterotrimeric GTP-binding protein and induces a marked increase in inositol 1,4,5-trisphosphate, leading to increases in intracellular calcium concentrations (2, 24). Rises in intracellular calcium have been shown to modulate bradykinin-induced mesangial cell contraction (3). In this regard, contraction of mesangial cells in vitro has been shown to activate specific signaling events leading to induction of early growth gene expression and activation of protein kinase C and S6 kinase (1, 17). Moreover, increases in intracellular calcium have been shown to activate MAPK via a calcium/calmodulin-dependent and -independent mechanisms (10). Whether the activation of MAPK we observed in mesangial cells in response to bradykinin stimulation is a result of mechanical forces leading to increases in intracellular calcium and, hence, activation of calmodulin is yet to be determined.
A number of recent observations have shown that bradykinin induces tyrosyl phosphorylation of proteins in a variety of cells (8, 25, 26). Our results demonstrate that bradykinin stimulates the tyrosyl phosphorylation of tubulin and MAPK in mesangial cells. The initiating cytoplasmic tyrosine kinase that promotes tyrosine phosphorylation of tubulin and MAPK in response to bradykinin is as yet undefined. However, our preliminary data in vascular smooth muscle cells indicate that bradykinin results in the activation and association of pp60-src with focal adhesion kinase and growth factor receptor binding protein-2 (40). Activation of pp60-src in response to growth factor stimulation results in its redistribution from the plasma membrane to the cytosol, where it may colocalize with microtubule-associated structures (21, 41). Recently it was shown that pp60-src phosphorylates tubulin in vivo (30). Tubulin is the main component of microtubules which form the mitotic spindle, and once phosphorylated, it is thought to play a regulatory role in cell growth and division (13, 30).
A large body of evidence indicates that MAPK plays a pivotal role in transmitting mitogenic signals in response to growth factors (4, 7). In the present study, we have shown that bradykinin induces tyrosyl phosphorylation and nuclear translocation of MAPK in mesangial cells, thus providing a link in the signal transduction pathway from the cytoplasm to the nucleus. Once in the nucleus, MAPK can phosphorylate and activate transcription factors such as TCF/ELK-1 resulting in c-Fos production, which in turn modulates transcription of other target proteins via AP-1 complex formation (12, 22).
The mechanisms through which G protein-coupled receptors like the bradykinin B2-receptor regulate MAPK are under intense investigation. MAPK activation by bradykinin B2-receptors may occur via a Ras-dependent or independent pathway by utilizing a pertussis toxin-sensitive or-insensitive G protein. In the case of receptors with intrinsic tyrosine kinase activity, several steps in the signal transduction leading to MAPK activation have been elucidated. Interaction of growth factors with their respective receptors activates intracellular tyrosine kinase domains, which result in the phosphorylation of Shc followed by its association with adaptor protein GRB2, which in turn promotes the interaction of guanine nucleotide exchange factor mSOS with Ras leading to its activation. Activated Ras in turn activates Raf-1, which in turn phosphorylates and activates MEK, ultimately leading to phosphorylation and activation of MAPK (36). Whether bradykinin activates MAPK via protein kinase C or by Ras activation or via the calcium/calmodulin pathway remains to be established.
The mechanism by which bradykinin induces nuclear translocation of MAPK is not clearly identified. Since MAPK lacks a canonical nuclear localization sequence, a number of alternative mechanisms have been proposed to explain this phenomenon (14). In addition, recent evidence indicates that the cytoskeletal microtubules may play a role in nuclear translocation of MAPK. First, MAPK was shown to be associated with cytoskeletal microtubules, and separation of the microtubule-bound MAPK from the cytoskeleton prevented its nuclear translocation in response to agonist stimulation (35). Second, stabilization of microtubules by taxol treatment prevented bradykinin-induced nuclear translocation of MAPK (20).
In summary, the findings of the present study demonstrate that bradykinin results in tyrosyl phosphorylation of a number of proteins in mesangial cells and suggest a role for tubulin and MAPK in the bradykinin signal transduction pathway leading to altered mesangial cell function. The significance of these findings to the in vivo actions of bradykinin on glomerular function is unclear at the present time. Since the in vivo actions of bradykinin on glomerular function are modulated by various autacoids such as nitric oxide and eicosanoids (38), the contribution of these autacoid systems on the signaling mechanisms initiated by bradykinin in mesangial cells needs to be addressed.
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ACKNOWLEDGEMENTS |
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We thank Kim Sutton for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46543, a Research Award from the American Diabetes Association (to A. A. Jaffa), a Merit Review Grant from the Research Service of the Department of Veterans Affairs (to R. K. Mayfield), a Postdoctoral Fellowship Award from the Medical University of South Carolina (to V. Velarde), National Heart, Lung, and Blood Institute Training Fellowship Grant HL-07260 (to P. S. Naidu), and a Medical Student Fellowship from the Pharmaceutical Manufacturers Association Foundation (to B. S. Miller).
Address for reprint requests: A. A. Jaffa, Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical Univ. of South Carolina, 171, Ashley Ave., Charleston, SC 29425.
Received 29 April 1997; accepted in final form 7 August 1997.
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