Activation of PKC-beta I in glomerular mesangial cells is associated with specific NF-kappa B subunit translocation

Anoop Kumar, Karen S. Hawkins, Meredith A. Hannan, and Michael B. Ganz

Division of Nephrology, Veterans Affairs Medical Center, and Section of Nephrology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in expression and activity of protein kinase C (PKC) isoforms and early transcription factors may account for alterations in cell behavior seen in diabetes. We studied the expression of PKC-beta I in rat glomerular mesangial cells (MCs) cultured in normal or high glucose and compared it with the temporal and spatial expression of dimeric transcription factor (NF-kappa B) p50 and p65. The results show that in unstimulated cells PKC-beta I and NF-kappa B p50 are distributed in the cytosol and, on stimulation, their distribution is perinuclear and they are localized to the membrane. Serum-starved MCs cultured in high-glucose medium exhibit a predominantly cytosolic localization of PKC-beta I and both p50 and p65 NF-kappa B. However, phorbol 12-myristate 13-acetate (PMA) stimulation of cells grown in the presence of high glucose resulted in membrane translocation of PKC-beta I that was associated with nuclear translocation of NF-kappa B p65, but not NF-kappa B p50. Moreover, the translocation to the nucleus for NF-kappa B p65 was significantly higher in MCs exposed to high glucose compared with those exposed to normal glucose. These observations indicate that the NF-kappa B p65, but not NF-kappa B p50, expression and translocation pattern mirrors that of PKC-beta I, which may be one important pathway by which signaling is enhanced in the high-glucose state.

protein kinase C; transcriptional factors; diabetes; nephropathy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HYPERGLYCEMIA HAS BEEN PROPOSED to be the main determinant in the initiation and progression of diabetic microvascular complications including nephropathy in both insulin-dependent and non-insulin-dependent diabetes mellitus (38, 40). The renal glomerular lesion in diabetes is characterized by glomerular hypertrophy that is followed by the deposition of extracellular matrix (1). Activation of protein kinase C (PKC) is one of the major mechanisms involved in high-glucose-induced glomerular injury, reactive oxygen production, and subsequent lipid peroxidation (18, 19, 29). Activation of specific PKC isoforms has also been shown to be associated with translocation to different subcellular compartments (11, 42). After cellular stimulation, PKC isoforms are targeted to distinct subcellular loci, including plasma membrane, nucleus, and cytoskeleton, suggesting specific biological roles (11, 17, 26, 39, 42). A preferential activation of PKC-beta isoforms in glomeruli isolated from diabetic rats has also been reported (27). Substantial evidence suggests that the accumulation of extracellular matrix in diabetes is related to changes in glomerular mesangial cell (MC) phenotypic behavior (1, 32, 36). Several studies support a role for PKC in high-glucose-induced early signaling events causing increased extracellular matrix accumulation by MCs (1, 4, 37).

A large number of genes that play important roles in immune and stress responses, inflammation, and apoptosis are regulated by the dimeric transcription factor nuclear factor (NF)-kappa B (5, 6, 9). The oxidative stress seen in diabetes and hyperglycemia appears to cause increased expression of various genes that are NF-kappa B dependent (8, 35). Hyperglycemia also activates NF-kappa B, in part by activation of PKC (28, 31). We sought to determine whether expression of PKC-beta I in rat MCs correlates with a temporal and spatial change in the expression of specific NF-kappa B isoforms. Unstimulated MCs exhibited similar expression of PKC-beta I and NF-kappa B p65 and p50 regardless of the glucose medium in which the cells were grown. On stimulation [phorbol 12-myristate 13-acetate (PMA)] PKC-beta I translocated to the membrane that was temporally associated with nuclear translocation of NF-kappa B p65 but not NF-kappa B p50. Moreover, the nuclear translocation of NF-kappa B p65 was more profound in high glucose. These results indicate that PKC activation leads to a preferential activation of NF-kappa B p65 that may be responsible for the activation of other genes in hyperglycemic conditions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and culture of MCs. MCs were isolated from the glomeruli of young adult male Sprague-Dawley rat kidneys according to a previously described protocol (15). The protocol for harvesting the MCs was approved by the Institutional Animal Care and Use Committee. Briefly, glomeruli were washed twice with Hanks' balanced salt solution (HBSS) and were plated onto 75-cm2 tissue culture flasks. The culture media contained DMEM with 5.6 mM glucose, 110 mg/ml sodium pyruvate, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 20% fetal bovine serum (FBS). Routine identification of MCs was performed by indirect immunofluorescence microscopy using rabbit IgG directed against vascular smooth muscle myosin and mouse anti-rabbit FITC-conjugated IgG. Cells showed uniformly strong positive staining of longitudinal filaments, a pattern that is characteristic of MCs (34).

Confluent passaged MCs were subcultured onto 60-mm petri dishes for further experiments. The passaged MCs (between passages 6 and 12) were grown in DMEM with either 5.6 (normal) or 30 mM (high) glucose. When the cells reached ~70% confluence, the medium was replaced with serum-free medium (0.5% FBS) with all other constituents, and the cells were grown for another 48 h. At the end of serum starvation, the cells in the PMA-treatment group were incubated with 0.1 µM PMA for 10 min, and both groups of cells were fractionated as described below.

Cell fractionation. The cells were fractionated as previously described with few modifications (16). Briefly, cells were washed twice with ice-cold PBS, harvested by scraping in 1 ml of ice-cold PBS containing protease inhibitors (100 µM PMSF, 1 µM aprotinin, and 1 µM pepstatin) into prechilled Eppendorf tubes and pelleted at 4°C. Cell pellets were resuspended in 100 µl of ice-cold homogenization buffer (60 mM Tris, pH 7.0, 0.25 M sucrose, 2 mM EDTA, and 1 mM beta -mercaptoethanol) containing protease inhibitors (100 µM PMSF, 1 µM aprotinin, and 1 µM pepstatin). The cell suspension was then subjected to three 30-s bursts of sonication, and cell lysis was checked under a microscope. The homogenates were centrifuged at 12,000 g for 20 min at 4°C. The supernatants were collected as cytosol fractions. An additional washing step with PBS (containing protease inhibitors) was included to eliminate the possibility of carryover of proteins from one fraction to another. The pellets were washed once with PBS (containing protease inhibitors described above) and resuspended in 500 µl of PBS (with protease inhibitors) and centrifuged as above. The supernatants were discarded, and the pellets were resuspended in 100 µl of homogenization buffer containing 1% Triton X-100 and incubated on ice for 30 min with intermittent vortexing. The samples were then centrifuged in a Beckman L8-60M ultracentrifuge at 100,000 g for 30 min at 4°C. The supernatants were collected as the membrane fractions. The pellets, which contain mainly the cytoskeleton and nucleus, were washed and resuspended in PBS (with protease inhibitors) and subjected to centrifugation as described above. The supernatants were discarded, and the pellets were resuspended in 100 µl of homogenization buffer containing 1% SDS, boiled for 10 min, and briefly microfuged to separate debris, and the supernatant was then collected as the particulate fraction (21). The protein concentrations were measured using the Micro BCA assay (Pierce, Rockford, IL).

Western blot analysis of PKC-beta I and NF-kappa B. Equal amounts of protein samples along with appropriate molecular size standards (Bio-Rad) were run on 10% SDS-PAGE under reducing conditions in a Mini Protean II Dual Slab Cell (Bio-Rad). Proteins separated on SDS gels were then transferred onto nitrocellulose membranes using a Mini Transblot electrophoretic transfer cell (Bio-Rad). The membrane was incubated in a blocking buffer (PBS, 0.05% Tween 20, 5% nonfat dry milk; 1% BSA) for 1 h at room temperature and then reacted with primary antibodies to PKC-beta I, NF-kappa B p50, or NF-kappa B p65 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,000 dilution) in the blocking buffer for 3 h at room temperature. This was followed by three washes with PBS-0.05% Tween 20 (PBS-Tween), incubation with horseradish peroxidase-conjugated secondary antibodies (1:4,000 dilution) in blocking buffer for 1 h at room temperature, and three additional washes with PBS-Tween. The reaction was detected using enhanced chemiluminescence (NEN Life Sciences Products, Boston, MA). Rat brain lysate was used as a positive control to identify PKC-beta I. The specificity of bands for NF-kappa B p50 or NF-kappa B p65 was tested using blocking peptides (Santa Cruz Biotechnology) specific for the respective antibodies by preadsorption with the blocking peptides. Translocation was determined by a comparison of the respective bands in the cytosol, membrane and particulate fractions, and densitometric analysis using Scion Image, a Windows-compatible version of National Institutes of Health Image software. Statistical analysis was done using the Statview program (SAS Institute, Cary, NC).

Immunofluorescence labeling for PKC-beta I and NF-kappa B. MCs cultured on chamber slides were serum starved as described above. The cells in the PMA-treatment group were stimulated for 10 min at 37°C with 0.1 µM of PMA in 400 µl of serum-free medium. The control (unstimulated) group received the same amount of medium. The wells were washed three times with PBS and fixed in 3.7% formaldehyde in PBS for 20 min at room temperature. The slides were then rinsed three times with PBS, and the nonspecific binding sites were blocked with 5% BSA in PBS for 60 min at room temperature. The cells were incubated for 1 h at room temperature with rabbit anti-PKC-beta I-specific or goat anti-NF-kappa B p50- or NF-kappa B p65-specific antibodies (1:100) in 0.1% BSA in PBS. This was followed by three washes with PBS. Labeling was achieved using affinity-purified FITC or rhodamine-conjugated secondary antibodies (1:200) by incubation in the dark for 1 h at room temperature as previously described (34). Applying the different primary antibodies simultaneously, we were able to perform double immunofluorescence. After being washed, cells were incubated with a mixture of FITC or rhodamine-conjugated secondary antibodies. Immunostaining was studied using a fluorescence microscope (Eclipse E600, Nikon). Images were collected separately and then merged using Scion Image.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Distribution of PKC-beta I. The expression pattern of PKC-beta I in different fractions as determined by Western blotting is represented in Fig. 1. In both the cytosol and membrane fractions, a single band at 80 kDa was observed, which comigrated with the rat brain positive control. In unstimulated cells, PKC-beta I was found predominantly in the cytosol fraction of cells in either the normal- or high-glucose concentration condition (96.7% in normal vs. 98.7% in high glucose; Fig. 2). In MCs cultured under normal glucose, stimulation with PMA for 10 min resulted in the rapid translocation of PKC-beta I to the membrane fraction with a substantial reduction in PKC-beta I that was present in the cytosol. PMA treatment for 60 min resulted in near complete activation of PKC-beta I; most PKC-beta I translocated from the cytosol to the membrane (Figs. 1 and 2). In MCs subcultured under high-glucose conditions, a similar pattern was evident; there was an increase in association with the membrane fraction by 10 min and an even greater increase by 60 min (Figs. 1 and 2). Under both glucose concentrations, PKC-beta I did not translocate to the particulate (nuclear) fraction.


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Fig. 1.   Representative Western blot analysis of subcellular fractions of mesangial cells (MCs) stained for protein kinase C (PKC)-beta I. Subcellular fractions were prepared from MCs without any treatment (unstimulated) or after stimulation with phorbol 12-myristate 13-acetate (PMA) for 10 or 60 min. Each lane was loaded with 50 µg of total protein.



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Fig. 2.   Expression of PKC-beta I in MC fractions. Western blots were prepared from subcellular fractions from MCs without any treatment (untreated) or after treatment with PMA for 60 min. The results represent the percentages of total PKC-beta I in unstimulated MCs grown in 5.6 mM glucose.

Representative immunofluorescence images of MCs labeled with primary antibody to PKC-beta I are shown in Fig. 3. In unstimulated MCs grown in both normal and high glucose, PKC-beta I was distributed throughout the cytosol. When the cells were exposed to PMA, PKC-beta I translocated to perinuclear and plasma membrane locations. PKC-beta I localization was similar in MCs grown in high glucose; beta I in unstimulated cells was distributed throughout the cytosol, with an intense staining and a punctate appearance. On exposure to PMA, PKC-beta I assumed a perinuclear and plasma membrane distribution. In both glucose concentrations, nuclei were not stained, as shown in Western blot analysis.


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Fig. 3.   Representative immunofluorescence photomicrographs of MCs demonstrating PKC-beta I. Immunofluorescence was performed on MCs grown in normal or high glucose that were either unstimulated (left) or PMA stimulated (right) for 10 min and stained for PKC-beta I.

Distribution of NF-kappa B. The distribution pattern of NF-kB p50 and p65 as determined by Western blotting is shown in Fig. 4. NF-kappa B p50 was present predominantly in the cytosol of MCs in both normal and high-glucose concentrations (85.9% in 5.6 mM vs. 86.2% in 30 mM). NF-kappa B p50 in both unstimulated and stimulated and low and high glucose remained predominantly in the cytosol fraction. NF-kappa B p50 was not visibly evident in the particulate fraction (1.5% in normal glucose vs. negligible amount in high glucose) (Figs. 4 and 5A). However, NF-kappa B p65 exhibited significantly more particulate localization in unstimulated cells in both glucose concentrations (Figs. 4 and 5B), in addition to cytosolic localization. On PMA stimulation, the amount of p65 increased significantly in the particulate fraction of cells in 30 mM glucose compared with cells exposed to 5.6 mM glucose (Fig. 5B; 35.4 vs. 20.4%).


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Fig. 4.   Western blot analysis of MC subcellular fractions stained for the dimeric transcription factor nuclear factor (NF)-kappa B p50 and NF-kappa B p65 (top and bottom, respectively). Subcellular fractions were the same fractions that were used for the determination of PKC-beta I. Each lane was loaded with 25 µg of total protein.



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Fig. 5.   A: expression of NF-kappa B p50 in particulate fractions of MCs. Densitometric readings of Western blots of particulate fractions of MCs grown in 5.6 and 30 mM glucose are shown. The results represent the percentages of total NF-kappa B p50 in unstimulated MCs grown in 5.6 mM glucose. B: expression of NF-kappa B p65 in particulate fractions of MCs. Densitometric readings of Western blots of particulate fractions of MCs grown in 5.6 and 30 mM glucose are shown. The results represent the percentages of total NF-kappa B p65 in unstimulated MCs grown in 5.6 mM glucose.

Immunostaining for PKC-beta I and NF-kappa B. In data not shown, on PMA exposure p65 showed redistribution into nuclear and perinuclear areas in a fashion similar to that shown in Western blots. We performed double immunostaining using two antibodies (immunolabeled with FITC and/or rhodamine) to determine colocalization of PKC-beta I and NF-kappa B p50 and NF-kappa B p65. The double-staining pattern for PKC-beta I and NF-kappa B p50, as shown in Fig. 6, closely resembles the pattern seen with single immunostaining. The results show that in unstimulated cells PKC-beta I and NF-kappa B p50 are distributed in the cytosol, and, on stimulation, their distribution is perinuclear and on the membrane. The results shown in Fig. 6 also show that PKC-beta I and NF-kappa B p65 are distributed in the cytosolic and membrane areas. However, MCs in both glucose concentrations show nuclear staining for p65, which is similar to the results in Western blots. On stimulation, the cells showed intense staining in the nucleus and perinuclear areas, indicating that there is increased nuclear translocation of p65 correlating with the spatial perinuclear localization PKC-beta I.


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Fig. 6.   Representative photomicrographs of double staining of MCs [unstimulated (left) or PMA stimulated (right)] for PKC-beta I and NF-kappa B. Red staining represents the staining pattern for PKC-beta I, green staining represents that for NF-kappa B p50 or NF-kappa B p65, and yellow staining reveals the colocalization of proteins.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that PKC-beta I activation (translocation) was temporally associated with nuclear translocation of NF-kappa B p65 but not NF-kappa B p50. In addition, the temporal and spatial localization of NF-kappa B p65 was greatly accentuated when cells were grown under high-glucose conditions. These results may help to define the specific biological roles for isoforms of PKC and those of transcription factor NF-kappa B.

Several studies have shown that MCs express calcium-dependent classic isoforms of PKC such as alpha , beta I, beta II, and gamma  (16, 21, 33, 34). PKC-beta I and -beta II are the isoforms of PKC most frequently associated with alterations in MC phenotypic behavior such as proliferation and matrix deposition (41). Previous studies from our laboratory have shown that PKC-beta II is temporally associated with rapid MC proliferation during development (2, 34). With further renal maturation, this isoform becomes downregulated in MCs (34). Therefore, in the present investigation we concentrated on the PKC-beta I isoform, which is readily expressed in adult MCs.

High glucose has been found to induce numerous changes in the phenotypic behavior of various cells, including glomerular MCs (13, 20, 22, 30). Hyperglycemia-induced glomerular injury has been shown to modulate MC biological behavior, which is reflected in early changes in membrane transport (1, 16, 33, 37). Cellular distribution studies have demonstrated that PKC isoforms exhibit tissue- and region-specific expression in different cells (3, 14). These findings have led to the notion that different PKC isoforms may be activated by different agonists and/or at different intracellular sites, allowing for the selective activation of isoforms in response to external stimuli.

Several studies have shown that the activity of PKC is elevated in the retina, glomeruli, and heart of diabetic animals, in purified microvessels from diabetic animals, and in endothelial and mesangial cells cultured in high glucose or galactose (4, 14, 21, 23, 24, 29, 42). Our data in the present study showing that the expression of PKC-beta I is enhanced in the presence of high glucose, and PMA stimulation causes membrane translocation of these PKC isoforms, are consistent with previous observations (21, 25, 42). However, some studies have also demonstrated that exposure of MCs to high glucose for 24-48 h in the presence of PMA results in translocation of PKC isoforms to membrane and nuclear and/or cytoskeletal compartments (12, 21).

Our observations in the present study also indicate that enhanced expression of PKC isoforms is associated with nuclear translocation of NF-kappa B p65. NF-kappa B is a major target of intracellularly induced oxidative stress (8, 35). The proteins of the NF-kappa B family form the inactive heterodimeric complexes in the cytoplasm of cells. Activation of NF-kappa B results from the dissociation of these inactive complexes from their inhibitory proteins and translocation of the active NF-kappa B complexes to the nucleus (5-7, 10). Such phenomena lead to changes in vascular homeostasis and endothelial dysfunction manifested in diabetic vascular complications, such as angiopathy, neuropathy, and nephropathy (8). Unlike p65, NF-kappa B p50 does not translocate simultaneously to the nucleus. It is therefore conceivable that signaling that leads to changes in MC behavior is achieved through a PKC-beta I and NF-kappa B p65 pathway. These results indicate the possibility of a dissociation of NF-kappa B p50 from the p50-p65 heterodimeric complex during PKC activation after stimulation of MCs.

In summary, our results indicate that MCs cultured in high-glucose medium exhibit enhanced membrane translocation of PKC-beta I on stimulation with PMA. This was associated with nuclear translocation of NF-kappa B p65. These observations may enhance our understanding of intracellular events associated with MC susceptibility to injury and subsequent hyperactivity associated with diabetes.


    ACKNOWLEDGEMENTS

This work was supported by American Heart Established Investigator Grant 96001450 and a Veterans Affairs Merit Review (to M. B. Ganz).


    FOOTNOTES

Address for reprint requests and other correspondence: A. Kumar, Section of Nephrology, Research Service 151 (W), Cleveland VA Medical Center, 10701 East Blvd., Cleveland, OH 44106 (E-mail: axk38{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 March 2001; accepted in final form 17 May 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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