LDL stimulates collagen mRNA synthesis in mesangial cells through induction of PKC and TGF-beta expression

Hyun Soon Lee, Bong Cho Kim, Hye Kyung Hong, and Young Sook Kim

Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Abnormal lipid accumulation in glomeruli could be implicated in the pathogenesis of glomerulosclerosis. Low-density lipoprotein (LDL) stimulates collagen mRNA expression in cultured human mesangial cells (HMC). To explore the possible molecular mechanisms by which LDL promotes collagen gene expression, we examined the effects of LDL on protein kinase C (PKC) activity and transforming growth factor-beta (TGF-beta ) expression in relation to collagen gene regulation in HMC. LDL (200 µg/ml) induced an acute increase in PKC activity, particularly PKC-alpha and -delta , within 15 min, which decreased to control value at 2 h. LDL stimulated TGF-beta 1, and alpha 1(I) and alpha 1(IV) collagen mRNA expression within 30 min of incubation with HMC, and levels remained elevated until hour 4. LDL induced the secretion of TGF-beta by HMC. This TGF-beta was shown by CCL-64 mink lung cell assay to be, in part, bioactive. The stimulatory effects of LDL on collagen gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X (GFX) or the downregulation of PKC using phorbol myristate acetate. Neutralizing antibody to TGF-beta inhibited the increased collagen mRNA expression by HMC exposed to LDL. The downregulation or inhibition of PKC did not affect the stimulatory effect of LDL on TGF-beta mRNA or protein expression. These results suggest that in HMC, LDL stimulates collagen mRNA expression through the rapid activation of PKC-alpha and -delta and transcriptional upregulation of TGF-beta . Thus PKC and TGF-beta may function as independent key signaling intermediaries in the pathway by which LDL upregulates collagen gene expression in HMC.

glomerulosclerosis; lipids; transmembrane signaling; collagen gene


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ACCUMULATION of extracellular matrix proteins in the mesangial area is an important component of glomerulosclerosis. Glomerular mesangial cells, which are often compared with vascular smooth muscle cells (VSMC), are a major site of synthesis of matrix proteins in chronic glomerular disease.

Studies have suggested that lipid abnormalities may be involved in the progression of glomerular disease (reviewed in Ref. 15). Low-density lipoprotein (LDL) can upregulate collagen gene expression after incubation for 48 h with cultured human mesangial cells (HMC) (17). The stimulation by LDL of quiescent HMC, which in turn oversecretes matrix proteins, might require a number of coordinated cellular events. Information regarding a variety of extracellular signals is relayed across the cell membrane to regulate many intracellular signals via a signal-bifurcating pathway, protein kinase C (PKC) activation and Ca2+ mobilization (20).

PKC comprises of a family of 11 different gene products that are classified into Ca2+-dependent or conventional PKC (alpha , beta , and gamma ) or Ca2+-independent or novel PKC (delta , epsilon , eta , theta , and µ) and atypical PKC groups according to their structure and function (21). Some experiments have shown that in cultured VSMC, LDL activates phosphoinositide turnover and PKC and that these responses are mediated by LDL receptor (4, 23, 26). Other investigators, however, have argued that LDL elevates intracellular calcium and pH but not phosphoinositide turnover in VSMC and that these responses are not mediated by LDL receptor (25). In HMC, specific receptor-mediated endocytosis of LDL has been clearly demonstrated (10, 18). In cultured rat mesangial cells (RMC), PKC activity was acutely and transiently activated by LDL, and more prolonged exposure of cells to LDL increased both transforming growth factor-beta (TGF-beta ) bioactivity and fibronectin synthesis (28).

TGF-beta exists in mammals in three isoforms (beta 1, beta 2, and beta 3), and its overproduction has been implicated in the pathogenesis of glomerulosclerosis (16). The neutralization of TGF-beta activity by antiserum against TGF-beta or the inhibition of TGF-beta expression by antisense oligonucleotides suppressed progression to glomerulosclerosis (1, 5). It has been suggested that TGF-beta increases mRNA levels in most matrix proteins in cultured cells (22), and in mouse mesangial cells, TGF-beta stimulates collagen and fibronectin synthesis (19). It has been shown that RMC express TGF-beta mRNA and secrete TGF-beta proteins, mostly in latent form (13). The induction of TGF-beta protein and its mRNA expression in RMC seems to be mediated through a tyrosine kinase-dependent mechanism but not through PKC (11). Studer et al. (28), however, suggested that the activation of PKC signals increased TGF-beta bioactivity.

The present study was designed to investigate the effects of LDL on PKC activity and TGF-beta in relation to collagen gene regulation in HMC. We found that LDL stimulated PKC, particularly PKC-alpha and -delta , and TGF-beta 1 expression at earlier time points in HMC, and these increases were intimately linked to the onset of enhanced collagen mRNA expression.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents. A PKC assay kit was obtained from Upstate Biotechnology, Lake Placid, NY. Polyclonal rabbit anti-PKC-alpha and -beta 1 were purchased from Santa Cruz Biotech (Santa Cruz, CA). Monoclonal mouse anti-PKC-delta was from Transduction Laboratories (Lexington, KY). Phorbol myristate acetate (PMA), protease inhibitors, and 10-nm gold-labeled goat anti-rabbit IgG was from Sigma Chemical (St. Louis, MO). GF-109203X (GFX) was obtained from Calbiochem (San Diego, CA), and [gamma -32P]ATP was from Amersham (Arlington Heights, IL). Monoclonal mouse anti-TGF-beta 1-beta 2-beta 3 was from Genzyme Diagnostics (Cambridge, MA), and polyclonal rabbit anti-TGF-beta was from R & D (Minneapolis, MN). TGF-beta 1 enzyme-linked immunosorbent assay (ELISA) kit was from Promega (Madison, WI). Other reagents were from the sources previously reported (17).

Culture of HMC. HMC were obtained from adult nephrectomy specimens, as previously described (18). Culture medium was made of DMEM supplemented with 20% fetal calf serum, 200 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml, and amphotericin 0.25 µg/ml). For the present experiments, cells between the 5th and 7th passage were used.

LDL isolation. Human LDL (density, 1.019-1.063) and lipoprotein-deficient serum (density >1.215) were isolated from the plasma of normal volunteers by the method of sequential ultracentrifugation, as previously described (18). Isolated LDL was dialyzed for 24 h at 4°C against buffer a containing 0.15 M NaCl and 0.24 mM EDTA, pH 7.4. After dialysis, LDL was stored at 4°C under nitrogen and was used within 14 days.

Experimental conditions. HMC were grown to near confluence. The cells were synchronized to quiescence in serum-free DMEM containing 5 µg/ml insulin-transferrin-selenite for 48 h. After synchronization, experiments were performed by the addition of 200 µg LDL protein per ml serum-free DMEM to HMC for 0.25, 0.5, 1, 2, and 4 h at 37°C. In some experiments, 2 µM of GFX, a potent PKC inhibitor (29), or 0.5 µM of PMA was added to cells 30 min or 24 h prior to LDL administration. In a given experiment, simultaneous control monolayers were treated with serum-free DMEM alone.

Assay of PKC activity. Quiescent HMC in 6-well plates were exposed to 200 µg/ml LDL for the required time intervals, and PKC activity was assayed using QKRPSQRSKYL as a specific substrate peptide as described by manufacturer (Upstate Biotech). Cells were washed rapidly three times with 25 mM Tris-buffered saline (TBS) at 4°C. After 1 ml of extraction buffer [PKB: containing 25 mM Tris · HCl, pH 7.4, 2 mM EGTA, 10 mM beta -mercaptoethanol, 20 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] was added to each well, cells were harvested and homogenized. Aliquots of the homogenate were taken to measure total PKC activity. For PKC assay, 10 µl of sample was added to a microcentrifuge tube, where the same amounts of substrate cocktail, inhibitor cocktail, and lipid activator were present. After the addition of 10 µl diluted [gamma -32P]ATP mixture, the microcentrifuge tube was incubated at 30°C for 10 min, and a 25-µl aliquot was transferred onto the center of numbered P81 phosphocellulose paper. Assay squares were washed in 40 ml 0.75% phosphoric acid and then in 20 ml acetone, then transferred to a scintillation vial. After scintillation cocktail was added, radioactivity was determined by a scintillation counter. Controls for endogenous phosphorylation of proteins in the sample extract were performed by substituting assay dilution buffer for substrate cocktail.

PKC specific activity (pmol phosphate incorporated into substrate peptide/min) was calculated as follows
<FR><NU>(PKC activity − enzyme background) × 2.4</NU><DE>specific radioactivity × 10 min</DE></FR>
PKC data were expressed as density units, calculated as the ratio of PKC specific activity of the sample to that of normal control.

Cell fractionation and PKC immunoblots. Quiescent HMC in 100-mm plates were stimulated with 200 µg/ml LDL for 15 min or 30 min. Cells were washed twice with ice-cold PBS. After 100 µl of buffer A containing 20 mM Tris · HCl, pH 7.5, 5 mM EDTA, 1 mM PMSF, and 10 µg/ml leupeptin was added, cells were harvested and sonicated. The homogenates were centrifuged at 32,000 rpm for 1 h at 4°C and the supernatants were retained as the cytosolic fraction. The pellets were resuspended in 100 µl of buffer B (buffer A with 1% Triton X-100), sonicated, left on ice for 1 h, and centrifuged again at 32,000 rpm for 1 h at 4°C. The supernatants were collected and used as the membrane fraction. Protein concentration was determined using a BCA protein assay reagent (Pierce, Rockford, IL). Aliquots were then electrophoretically resolved in 10% polyacrylamide gel in SDS buffer and transferred onto nitrocellulose membranes as we have previously described (17). The blots were incubated with rabbit anti-PKC-alpha (1:1,000) or -beta 1 (1:500) or mouse anti-PKC-delta (1:250) at 4°C overnight. Bound primary antibody was visualized by the subsequent incubation with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence system kit (Amersham). Densitometry was performed using an imaging densitometer (Bio-Rad model GS-700). The specificity of primary antibody reaction with sample was eliminated in the presence of peptide that was used to generate the antibody.

Preparation of conditioned media and activation of latent TGF-beta . At the end of the experiment, the culture supernatants from HMC were harvested in siliconized tubes in ice, centrifuged to remove cell debris, and subjected to bioassay. To activate latent TGF-beta , an aliquot of medium was diluted to adjust protein concentration to 50-100 µg/ml and heated to 80°C for 8 min. Samples were analyzed both before heating (to measure the active form) and after heat activation of the latent form (to determine total TGF-beta ).

Quantitation of secreted TGF-beta 1. The concentration of TGF-beta 1 in conditioned media was determined using a TGF-beta 1 ELISA kit, according to the manufacturer's instructions (Promega). Briefly, samples were activated with 1 N HCl for 15 min at room temperature followed by neutralization with 1 N NaOH. Samples were added to microtiter plates, which were coated with monoclonal anti-TGF-beta 1. After incubation for 90 min at room temperature, the plates were incubated with rabbit anti-human TGF-beta 1 antibody for 2 h. Then goat anti-rabbit IgG conjugated to horseradish peroxidase was added and incubated for 2 h. The assay was developed with the peroxidase substrate for 3-4 min at room temperature followed by 1 M phosphoric acid. The absorbance was measured at 450 nm with a Molecular Devices (Spectra Max 250). Standard curves for TGF-beta 1 were prepared using serial dilutions of exogenous TGF-beta 1 (Promega).

Bioassay of TGF-beta activity. The biological activity of TGF-beta was assayed using a modification of method of Danielpour et al. (8). In brief, mink lung epithelial cells (American Type Culture Collection CCL-64) were plated onto 24-well plates in DMEM containing 0.2% lipoprotein-deficient serum. After 24 h, test samples were added, and 22 h later, cells were exposed to 0.5 µCi of [3H]thymidine per well in a total of 0.5 ml/well for 2 h at 37°C. Cells were then washed, treated with 0.5% trypsin/EDTA, harvested, and radioactivity was measured using a Canberra scintillation counter. Data were expressed as the percentage of thymidine incorporation in untreated control mink lung cells.

Immunogold electron microscopy. Synchronized mesangial cells in 35-mm tissue culture dishes were incubated with 200 µg/ml LDL or serum-free DMEM for 2 h. After two washes with 0.1 M PBS, cells were exposed to 0.5% trypsin EDTA for 20 min. Cells were scraped off the culture dish and spun down. The pellet was immersed in 2% paraformaldehyde and 0.2% glutaraldehyde for 2 h at 4°C, washed twice for 5 min at 4°C with 0.1 M PBS, infiltrated with 2.3 M sucrose for more than 1 h, and frozen in liquid nitrogen. Ultrathin frozen sections were prepared using a Reichert FCS low-temperature sectioning system, and these were mounted on Formvar-coated 100-mesh nickel grids. These were treated with 6 M acid-urea for 30 min at 4°C to unmask a hidden epitope of TGF-beta and then incubated overnight with polyclonal rabbit anti-TGF-beta (1:10). Excess unbound primary antibody was washed off the grids using PBS containing 1% bovine serum albumin and 0.05% Tween-20, as we have previously described (31). The grids were then exposed to 10-nm gold-labeled goat anti-rabbit IgG (1:30) for 2 h at room temperature and washed first with PBS and then with distilled water. Afterward, the sections were stained for 10 min with uranyl acetate and for 5 min with lead citrate, and observed with a Hitachi 7100 electron microscope. Specificity for the TGF-beta staining was further confirmed by absorption test: anti-TGF-beta with a staining titer of 1:10 was diluted twofold in buffer and mixed with an equal volume of 1 µg/ml TGF-beta . After incubation at 37°C for 1 h, the mixture was centrifuged at 4°C at 9,000 rpm for 30 min, and the supernatant was tested for the ability to stain TGF-beta . For negative controls, the primary antibody was omitted or replaced by corresponding nonimmune serum.

Northern blot analysis. Total cellular RNA was isolated from confluent experimental and control cultures by the acid guanidium thiocyanate-phenol-chloroform extraction method. RNA samples were electrophoresed through 1.2% agarose-formaldehyde gels and transferred by capillary blotting onto nylon filters. A cDNA probe for the TGF-beta 1 detected a transcript of 2.1-kb EcoR I fragment (14) and was obtained from American Type Culture Collection (Rockville, MD). We used the cDNA probes for the alpha 1(IV) collagen and alpha 1(I) collagen as described (17). Using a Rediprime labeling kit, the random primer DNA labeling system, 10-50 ng of a cDNA template was radiolabeled with 50 µCi of [32P]dCTP. The blotted membranes were incubated for 12-24 h with the specific 32P-labeled cDNA probes in standard hybridization solution and subjected to high-stringency washes. The filters were dried and exposed at -70°C to Kodak X-OMAT-AR film with the aid of intensifying screens for 6-36 h. The mRNA levels for TGF-beta 1 and alpha 1(IV) and alpha 1(I) collagen were expressed as a ratio of the optical density units for either TGF-beta 1 or collagen to beta -actin.

Detection of type IV collagen. Type IV collagen in cytosolic fraction of HMC was detected by Western blot analysis as we have previously described (17).

Statistics. Results were expressed as means ± SD for three separate experiments. Results were analyzed by two-way ANOVA among three groups or by Wilcoxon's rank sum test between two groups. P < 0.05 was considered significant.


    RESULTS
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RESULTS
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LDL increases alpha 1(IV) and alpha 1(I) collagen mRNA levels. Starting 30 min after incubation with HMC, LDL at concentrations of 200 µg/ml stimulated mRNA expression of alpha 1(IV) and alpha 1(I) collagen; expression remained high until hour 4 (Fig. 1). At 1 h, LDL had increased the alpha 1(IV) and alpha 1(I) collagen transcripts to 2.2- and 6.2 times the levels in the control cells. At 2 h, the level of alpha 1(IV) collagen mRNA in HMC exposed to LDL had increased to 2.8-fold higher than in the control cells. LDL also led to an increase in type IV collagen expression as estimated by immunoblotting compared with controls (data not shown).



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Fig. 1.   Northern blot analysis of alpha 1(IV) collagen and alpha 1(I) collagen mRNA in human mesangial cells (HMC). Cells were incubated with serum-free DMEM alone (lanes 1, 3, 5, and 7) or with addition of 200 µg/ml low-density lipoprotein (LDL, lanes 2, 4, 6, and 8) for 30 min (lanes 1 and 2), 1 h (lanes 3 and 4), 2 h (lanes 5 and 6), or 4 h (lanes 7 and 8). Blots were hybridized with [32P]-labeled cDNAs for alpha 1(IV) collagen (A), alpha 1(I) collagen (B), and beta -actin (C). D: quantitative expression of alpha 1(IV) and alpha 1(I) collagen mRNA abundance after correcting for the beta -actin signal. The alpha 1(IV) and alpha 1(I) collagen mRNA levels of treated HMC are expressed as % increases above the mRNA levels of untreated controls. Values are presented as the means ± SD of 3 separate experiments.

LDL stimulates PKC activity. When HMC were exposed to LDL, total PKC activity 15 min after the addition of LDL had increased to 1.9 times the control level, although at 2 h, this effect had disappeared. The time-dependent effects of LDL on total PKC activity of HMC are shown in Fig. 2. Pretreatment of HMC with 0.5 µM PMA for 24 h prior to LDL administration resulted in the marked downregulation of PKC activity to less than 10% of control activity. The exposure of cells to the PKC inhibitor, GFX (2 µM), 30 min before LDL administration, also caused a significant reduction of PKC activity; this fell to 20% of its control value. The addition of anti-TGF-beta to LDL, however, did not affect the LDL-induced PKC activity.


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Fig. 2.   Effects of LDL on protein kinase C (PKC) activity of HMC. Data are expressed as density units, calculated as the ratio of PKC specific activity of the sample to that of normal control. Values are means ± SD of 3 comparable experiments. * P < 0.05

LDL did not affect PKC-alpha in the cytosolic fraction, but increased membrane content to 140 ± 19% and 196 ± 5% of basal, respectively, at 15 and 30 min. LDL caused a significant increase in cytosolic PKC-delta to 214 ± 20% and 311 ± 136% of basal, respectively, at 15 and at 30 min, accompanied by a significant increase in membrane content to 154 ± 4% and 210 ± 83% of basal, respectively (Fig. 3). PKC-beta 1 was not expressed at all in both control and LDL-treated cells.


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Fig. 3.   Effects of LDL on expression of PKC-alpha and -delta in HMC as monitored by Western blot analysis. Cells were incubated with serum-free DMEM alone (lane 1) or with addition of 200 µg/ml LDL for 15 min (lane 2) or 30 min (lane 3). PKC isoforms in cytosol and membrane fractions were separately measured.

LDL induces TGF-beta mRNA expression and the secretion of bioactive TGF-beta . Thirty minutes after the addition of LDL, mRNA levels of TGF-beta 1 had increased to 1.3 times their levels in the control cells. After 2 h of culture with LDL, the mRNA levels had increased to 1.6-fold compared with control cells. The magnitude of this effect persisted until hour 4 (Fig. 4).



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Fig. 4.   Northern blot analysis of transforming growth factor-beta 1 (TGF-beta 1) mRNA in HMC. Cells were incubated with serum-free DMEM alone (lane 1) or with addition of 200 µg/ml LDL for 30 min (lane 2), 1 h (lane 3), 2 h (lane 4), or 4 h (lane 5). Blots were hybridized with [32P]-labeled cDNAs for TGF-beta 1 (A) and beta -actin (B). C: quantitative expression of TGF-beta 1 mRNA abundance after correcting for the beta -actin signal. Data are expressed as described in Fig. 1 legend.

LDL caused a significant increase in TGF-beta 1 protein secretion by HMC as measured by ELISA to values 8.3-fold higher than control values on hour 0.5 and 9-fold higher on hours 2 and 4 (Fig. 5).


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Fig. 5.   LDL induces secretion of TGF-beta in HMC. Cells were incubated with serum-free DMEM alone (open bars), or with the addition of 200 µg/ml LDL (gray bars) for between 30 min and 4 h. Concentrations of TGF-beta were determined by using enzyme-linked immunosorbent assay with specific antibodies against TGF-beta 1. Data are representative of 3 different experiments, and values are means ± SD of triplicate determinations. * P < 0.05 vs. control.

Figure 6 shows the effects of LDL on TGF-beta bioactivity in culture media of HMC, as assessed by growth inhibition of CCL-64 mink lung cells. All conditioned media used in this assay were diluted to 1:15. Heat-activated conditioned medium (total TGF-beta ) obtained from control HMC (no LDL) restricted cell growth to 87% of untreated control cells, whereas unheated medium (active TGF-beta ) had no effect; this confirms the results of a previous report (13), which stated that normal mesangial cells secrete TGF-beta mostly in latent form. Unheated medium from HMC exposed to LDL for 2 h significantly inhibited the growth of CCL-64 mink lung cells compared with that from control HMC (65 ± 12% vs. 97 ± 6%, P < 0.01), suggesting that conditioned medium from HMC exposed to LDL contains biologically active TGF-beta . Heat-activated conditioned medium also significantly inhibited the growth of mink lung cells compared with that from control HMC (49 ± 12% vs. 87 ± 8%, P < 0.01). This inhibitory effect was completely reversed by the addition of monoclonal anti-TGF-beta (LDL + anti-TGF-beta ).


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Fig. 6.   Growth inhibition of CCL-64 mink lung cells by conditioned medium (before and after heat activation) of HMC. Culture supernatants of HMC exposed for 2 h to serum-free DMEM alone (no LDL), or with addition of 200 µg/ml LDL (LDL), or both LDL and anti-TGF-beta (LDL + anti-TGF-beta ) were incubated with mink lung cells. Data are expressed as percentage of [3H]thymidine incorporation in untreated control cells. Data are representative of 3 different experiments, and values are means ± SD of triplicate determinations. * P < 0.01 vs. cells treated with unheated conditioned medium without LDL. ** P < 0.01 vs. cells treated with heated conditioned medium without LDL.

By immunoelectron microscopy of ultrathin frozen sections, gold particle labeling against TGF-beta was shown mainly in the cytosol of cells incubated with LDL for 2 h (Fig. 7). In contrast, no gold particles were seen in control cells incubated with serum-free DMEM. Absorption test with anti-TGF-beta preabsorbed with TGF-beta gave negative results, indicating that the staining for TGF-beta was specific.


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Fig. 7.   Electron micrograph of gold-labeled antibody to TGF-beta in HMC incubated with serum-free DMEM alone (control, A) or with the addition of 200 µg/ml LDL (B) for 2 h. Distribution of gold particles is found in the cell incubated with LDL (B, inset), but not in control cell (A). N, nucleus. Magnifications: A, ×5,300; B, ×15,500; inset, ×46,500.

PKC inhibition or downregulation attenuates LDL-induced increase in collagen mRNA levels, but not TGF-beta 1 expression. The role played by PKC in LDL-induced increase in collagen gene expression was assessed by inhibiting enzymatic activity of the kinase in HMC. When the GFX-pretreated HMC were incubated with LDL for 2 h, the LDL-induced increases in alpha 1(IV) mRNA expression were significantly inhibited (Fig. 8A). Prior exposure of HMC to PMA for 24 h before LDL also blocked the stimulatory effect of LDL on alpha 1(IV) collagen mRNA expression (Fig. 8A). However, downregulation or inhibition of PKC by PMA or GFX did not affect the action of LDL on TGF-beta 1 mRNA expression (Fig. 8B); nor did it affect the LDL-induced TGF-beta 1 secretion in media as measured by ELISA.



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Fig. 8.   Northern blot analysis of alpha 1(IV) collagen and TGF-beta 1 mRNA in HMC incubated for 2 h with serum-free DMEM alone (lane 1), or with the addition of 200 µg/ml LDL (lane 2), or in HMC pretreated with phorbol myristate acetate (PMA, lane 3) or GF-109203X (GFX, lane 4) and subsequently exposed to LDL for 2 h. Blots were probed for alpha 1(IV) collagen (A), TGF-beta 1 (B), and beta -actin (C). D: quantitative expression of alpha 1(IV) mRNA abundance after correcting for beta -actin signal. Data are expressed as described in legend to Fig. 1. * P < 0.01 vs. control, LDL + PMA, or LDL + GFX.

Anti-TGF-beta attenuates LDL-induced increase in collagen mRNA levels. The effects of pan-specific anti-TGF-beta neutralizing antibody on increases in collagen mRNA expression in HMC cultured with LDL are shown in Fig. 9. When cells were incubated with anti-TGF-beta antibody (25 µg/ml) along with LDL for 2 h, the expected increase in alpha 1(I) collagen mRNA expression was abrogated. However, the same concentration of control rabbit IgG had no effect on LDL-induced increase in collagen mRNA levels. This suggests an intermediate role for TGF-beta in the effects of LDL.



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Fig. 9.   Northern blot analysis of alpha 1(I) collagen mRNA in HMC incubated for 2 h with serum-free DMEM alone (lane 1), or with addition of 200 µg/ml LDL (lane 2), or both LDL and IgG (lane 3), or both LDL and anti-TGF-beta (lane 4). Blots were probed for alpha 1(I) collagen (A) and beta -actin (B). C: quantitative expression of TGF-beta 1 mRNA abundance after correcting for beta -actin signal. Data are expressed as described in legend to Fig. 1. * P < 0.01 vs. control or LDL + anti-TGF-beta .


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The experiments detailed in this report demonstrate that LDL rapidly stimulates PKC-alpha and -delta and TGF-beta expression in HMC. LDL also increases mRNA levels of alpha 1(I) and alpha 1(IV) collagen at earlier time points. The LDL-induced upregulation of collagen gene expression was inhibited by downregulation or inhibition of PKC or by the application of anti-TGF-beta neutralizing antibody, suggesting that PKC and TGF-beta are intimately linked to the onset of enhanced collagen mRNA expression.

The activation of PKC was seen within 15 min of exposure of HMC to LDL and had disappeared at hour 2. LDL increased membrane-associated PKC-alpha and -delta and cytosolic PKC-delta but did not stimulate a change in PKC-alpha cytosol content. It has been frequently observed that, upon activation by agonists, PKC is translocated from the cytosolic to the membrane fraction of the cell (21, 26). The lack of LDL-induced translocation of PKC-alpha and -delta in the present study suggests that cellular compartmental analysis by immunoblot alone may not be sufficient to detect translocation of PKC. The physiological significance of translocation, however, is not clear. Nishizuka (20, 21) suggested that the translocation of PKC could be a biochemical artifact caused by the presence of large quantities of Ca2+ chelators to prevent the proteolysis during the homogenization. It is tempting to speculate that LDL activates PKC via a specific LDL receptor on the plasma membrane of HMC. Lipid peroxidation products increased measurably after LDL was incubated with HMC for 1 h (17). Thus the return of PKC activity to control levels at 2 h might partly be related to gradual lipid peroxidation of LDL by HMC, with reduced binding affinity to the LDL receptor.

The prolonged exposure of HMC to PMA prior to LDL administration resulted in the downregulation of PKC and the subsequent abolition of the stimulatory effects of LDL on collagen gene expression. Inhibition of PKC by the addition of GFX to HMC 30 min prior to LDL administration also caused a marked reduction in alpha 1(I) and alpha 1(IV) mRNA stimulation by LDL. Thus the rapid activation of PKC by LDL seems to signal the upregulation of collagen gene expression in HMC. In cultured human umbilical vein endothelial cells, increased PKC activity also mediated the onset of enhanced collagen IV mRNA expression (6).

We also found that exposure of HMC to LDL activated TGF-beta mRNA synthesis and induced the secretion of bioactive TGF-beta within the first few hours of stimulation. A number of studies, including our own, have indicated that changes in TGF-beta mRNA levels in the glomeruli or in mesangial cells are associated with a change in protein expression (12, 24, 27). The degree of TGF-beta protein expression was much greater than that of mRNA expression as shown by us and others (24), suggesting that the synthesis rate of TGF-beta protein is fast or its degradation rate is slow or both. In addition, some researchers observed, as we did, that TGF-beta mRNA levels could reflect TGF-beta bioactivity (12, 24, 27), whereas others negated that possibility (2, 7).

Two-hour incubation of HMC with neutralizing antibody to TGF-beta along with LDL inhibited both the expected increase in alpha 1(I) collagen mRNA expression and the secretion of biologically active TGF-beta . These observations indicate that anti-TGF-beta antibody effectively neutralized the biologically active TGF-beta secreted from activated HMC by LDL, abrogating the expected collagen gene upregulation. It has been demonstrated that in murine mesangial cells exposed to LDL or high glucose levels, anti-TGF-beta antibody prevents the expected increase in collagen or fibronectin synthesis (28, 32). In experimental glomerulonephritis, the administration of anti-TGF-beta antibody neutralized TGF-beta activity, thus resulting in decreased proteoglycan synthesis (5).

In the present study, the stimulatory effects of LDL on TGF-beta mRNA or protein expression were not blocked by inhibition or downregulation of PKC, suggesting that LDL stimulates TGF-beta mRNA expression not mediated through PKC activation. In support of our observation, Hirakata et al. (11) demonstrated that PKC is not involved in the induction of TGF-beta mRNA expression in RMC. Furthermore, the antimitotic effect of TGF-beta in RMC and VSMC was not affected by downregulation of PKC (3, 30). Others, however, have suggested that the activation of PKC signals increases in TGF-beta mRNA expression (9) or TGF-beta bioactivity (28). As suggested by Kaname et al. (13), the induction of TGF-beta mRNA expression in RMC may be mediated through either a PKC-independent or -dependent pathway, and this requires further confirmation.

Despite an early and transient rise in PKC activity, an LDL-induced increase in TGF-beta and collagen mRNA levels was sustained over 4 h in the present study. Thus TGF-beta could be involved not only in the onset of collagen gene upregulation by LDL but also in its maintenance in HMC. LDL-induced enhanced collagen mRNA expression leads to increased collagen synthesis (17). In view of the rapid and/or transient induction of PKC, TGF-beta , and collagen expression, the in vitro model may not be representative of the in vivo situation of chronic progressive glomerulosclerosis. Nonetheless, the in vitro data could indirectly explain the potential mechanisms by which LDL stimulates mesangial matrix in vivo eventually associated with glomerulosclerosis.

In summary, LDL rapidly stimulates PKC-alpha and -delta and TGF-beta expression in HMC. The LDL-induced upregulation of collagen gene expression was inhibited by the downregulation or inhibition of PKC or by the application of anti-TGF-beta neutralizing antibody. These results suggest that LDL stimulates collagen mRNA expression through the activation of PKC-alpha and -delta and transcriptional upregulation of TGF-beta in HMC. This effect of LDL might have a pathophysiological function in the pathogenesis of progressive glomerulosclerosis.


    ACKNOWLEDGEMENTS

This study was supported by grants from the Ministry of Health and Welfare (Korea) and Seoul National University Hospital.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. S. Lee, Dept. of Pathology, Seoul National Univ. College of Medicine, Chongno-gu, Yongon-dong 28, Seoul 110-799, Korea (E-mail: hyunsoon{at}plaza.snu.ac.kr).

Received 20 October 1998; accepted in final form 18 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Physiol 277(3):F369-F376
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society