Serotonin [5-hydroxytryptamine (5-HT)], a decarboxylation derivative of the amino acid tryptophan, is an active molecule released mainly from activated platelets1. Recent reports have emphasized that the proximal renal tubules also produced a significant amount of 5-HT that may modulate renal phosphorus transport in a paracrine fashion2. 5-HT is also known to have several biological activities, for example, it acts as a neurotransmitter in the central and sympathetic nervous systems, a potent constrictor of smooth muscle cells, and an activator of platelets3. The plasma concentration of 5-HT is particularly high in diabetic patients4,5, suggesting that it may be involved in the development of diabetic complications (through arterial contraction and platelet aggregation), although there is no clear clinical evidence for its involvement in the pathogenesis of diabetic nephropathy. However, a recent study by Nebigil et al showed the presence of 5-HT2A receptor subtype on glomerular mesangial cells6, suggesting that a direct effect of 5-HT on mesangial cells through its receptor might be involved in the development of diabetic nephropathy.
Expansion of the glomerular mesangial lesion is one of the histological features of diabetic nephropathy7. The mesangial matrix is normally composed of various macromolecules, including fibronectin, laminin, and collagen8,9. Among these molecules, the metabolism of type IV collagen has been extensively examined in diabetic mesangial lesions. Mesangial cells are thought to play an important role in the metabolism of type IV collagen by modulating its synthesis and degradation8,9. In vitro experiments using rat mesangial cells have shown that high glucose concentrations10 and exogenous transforming growth factor- 
(TGF-)11,12 induce the secretion of type IV collagen. However, the mechanism of accumulation of type IV collagen in diabetic mesangial lesions remains to be elucidated.
In the present study, we examined the effect of 5-HT on the production of type IV collagen by human mesangial cells, and demonstrated that 5-HT stimulated type IV collagen secretion from human mesangial cells by activation of protein kinase C (PKC) with subsequent increase in active TGF-.
METHODS
Chemicals
Serotonin (5-hydroxytryptamine hydrochloride crystalline), ketanserin (5-HT2A receptor antagonist), and calphostin C (a PKC inhibitor) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Calphostin C was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was less than 0.1% in the culture medium which did not affect viability and growth of mesangial cells. Sarpogrelate hydrochloride (5-HT2A receptor antagonist) was a generous gift from Tokyo Tanabe Co., Ltd. (Tokyo, Japan). Ondansetron (5-HT3 receptor antagonist) was a generous gift from Sankyo Co., Ltd. (Tokyo, Japan). All other chemicals were of the best grade available from commercial sources.
Cells
The experimental protocol was approved by the Human Ethics Review Committee of our institution and a signed consent form was obtained from each patient. Human glomeruli were isolated according to the method of Gröne et al13, with minor modifications14,15. Briefly, the kidneys of patients with ureteral cancer were removed and the cortices of normal sections were cut and pressed through a series of sieves of decreasing pore size (250, 125 and 110 
m). After passing through the finest sieve, the collected tissue was rinsed twice with Hank's balanced salt solution (HBSS; Nissui Pharmaceutical Co., Tokyo, Japan) and centrifuged (3,000 
g) for 10 minutes. The pellet was resuspended in 5 ml of HBSS containing 750 U/ml collagenase (Sigma) and incubated for 30 minutes at 37°C. After incubation, digested glomeruli were washed five times with phosphate buffered saline (PBS) and the glomerular cores were cultured at 37°C in RPMI 1640 (Life Technologies, Inc., Grand Island, NY, USA) with 20% fetal calf serum (FCS), 100 U/ml penicillin, 10 g/ml streptomycin, 250 g/ml amphotericin B and ITS™ premix (Becton Dickinson, Bedford, MA, USA) in 75 cm2 plastic tissue culture flasks in a 5% CO2 incubator. Cultured human mesangial cells between the fifth and eighth passages formed a uniform cell population as evaluated by the following criteria: (1) spindle shape, (2) positive immunohistochemistry with antibody for 
-actin and vimentin, and negative for cytokeratin and Factor VIII, and (3) contraction in response to angiotensin II. Mesangial cells (5 104 cells/well) were plated onto 12-well plates and incubated in RPMI 1640 with 20% FCS, 100 U/ml penicillin, 10 g/ml streptomycin, 250 g/ml amphotericin B for 24 hours. Then, cells were incubated with 5-HT for 24 hours, 48 hours or 72 hours for determination of type IV collagen secretion and TGF- bioactivity. 5-HT is known to induce mesangial cell growth16. In a series of preliminary studies, mesangial cell growth was induced by 5-HT as confirmed by using [3H] thymidine incorporation and cell counting assays. Thus, the data of type IV collagen secretion and TGF- bioactivity were expressed on a cellular protein basis.
Enzyme-linked immunosorbent assay of type IV collagen
Type IV collagen was measured by enzyme-linked immunosorbent assay described by Rennard et al17 with minor modifications. In brief, 50 l type IV collagen standards or conditioned media from mesangial cells were incubated with 50 l of anti-type IV collagen IgG diluted with PBS containing 0.5% bovine serum albumin at 4°C for 16 hours. In the next step, 90 l of the reaction mixture was transferred to the well of 96 multi-well plate (Falcon; Becton Dickinson and Co., Lincoln Park, NJ, USA) precoated with type IV collagen (1 g type IV collagen/well in 0.02 mol/liter carbonate buffer, pH 9.6 at 4°C overnight) and the plate was incubated at 37°C for 60 minutes. After rinsing each well three times with a washing buffer (PBS with 0.1% Tween 20), 100 l anti-rabbit IgG conjugated with peroxidase (Tago, Inc., Burlingame, CA, USA) was added to the well, incubated at 37°C for 60 minutes and rinsed three times. The final reaction mixture (0.04 mg/dl o-phenylenediamine dihydrochloride and 0.06% H2O2 0.05 mol/liter citrate buffer, pH 5.0) was then added and incubated at room temperature for two minutes. The reaction was stopped by adding 50 l H2SO4 followed by measurement of absorbance at 490 nm.
TGF-
bioassay
The assay used in the present experiments was essentially similar to that described previously by Ikeda, Lioubin and Marquardt18. An aliquot of the medium was used to determine the active form of TGF- by the bioassay described below. To determine the total TGF- in the medium, the remaining portion was acidified to pH 2 with 1 mol/liter HCl for 30 minutes at room temperature, dialyzed for 24 hours at 4°C against RPMI 1640 and subjected to the bioassay described below.
Mvl Lu mink lung epithelial cells (CCL-64) were purchased from American Type Culture Collection and cultured in RPMI 1640 with 10% FCS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (medium A). Before the bioassay, cells were subcultured for two hours at 3.5 103 cells/well in a 96-well tissue culture plate in 50 l of medium A. In the next step, 50 l of the conditioned medium to be tested was added to each well and incubated for four days at 37°C. During the last 24 hours of incubation, cells in each well were chased with 10 l of 10 Ci/ml [3H] thymidine, and the radioactivity incorporated into the cells was counted in a liquid scintillation spectrophotometer. For each assay, a standard curve for TGF- was constructed by 0.01 to 5 ng/ml of a recombinant human TGF-18 with or without 5-HT. [3H] thymidine incorporation into CCL-64 cells was not affected by 5 10-5 mol/liter of 5-HT (data not shown), demonstrating that 5-HT in the conditioned medium of mesangial cells did not alter TGF- bioactivity. Under these assay conditions, the addition of 10 g/ml of a mouse anti-human TGF- antibody (Genzyme, Cambridge, MA, USA)19 could neutralize > 95% of the TGF--induced growth inhibition of CCL-64 cells.
Assay of protein kinase C activity
PKC activity in human mesangial cells was assayed by MESACUP Protein Kinase Assay Kit (Medical and Biological Laboratories, Nagoya, Japan). Human mesangial cells (1 107 cells/well) in 2 ml of serum-free RPMI 1640 medium were treated with 10-6 mol/liter of 5-HT for indicated time intervals. The cells were washed three times in ice-cold PBS and detached from the plates with rubber policeman. Cells were suspended in 1 ml of sample preparation buffer (in mmol/liter, 5 EDTA, 10 EGTA, 50 2-mercaptoethanol, 1 PMSF, 10 benzamidine and 50 Tris-HCl, pH 7.5), and sonicated for 30 seconds at 4°C by Sonifier (Branson Sonic Power Co., USA). Homogenates were centrifuged at 100,000 g for one hour at 4°C. The supernatant was discarded and the precipitates were resuspended in 1 ml of preparation buffer and used as the membrane fractions. PKC activity in the membrane fractions was measured as follows. Twelve microliters of membrane fractions (5 g) were added to 108 l of component buffer (final concentration of reaction mixture was 3 mmol/liter MgCl2, 0.1 mmol/liter ATP, 2 mmol/liter CaCl2, 50 g/ml phosphatidylserine, 0.5 mmol/liter EDTA, 1 mmol/liter EGTA, 5 mmol/liter 2-mercaptoethanol and 25 mmol/liter Tris-HCl, pH 7.0). In the next step, 100 l of this mixture was transferred to each well coated with PKC-specific peptide (RFARKGSLRQKNV) using a multichannel pipette. Each well was incubated at 25°C for five minutes and the reaction was terminated by the addition of 100 l of 20% H3PO4, followed by washing five times with 200 l of PBS. To each well we added 100 l of biotinylated anti-phosphorylated serine antibody (2B9) and the mixture was incubated at 25°C for 60 minutes, and washed five times with PBS. The mixture was further incubated at 25°C for 60 minutes after the addition of 100 l of peroxidase conjugated streptavidin to each well, followed by washing five times with PBS. After the addition of 100 l of H2O2 and o-phenylenediamine, the mixture was incubated at 25°C for five minutes, followed by the addition of 100 l of 20% H3PO4. Finally, the absorbance was read at 492 nm with a microplate reader. Nonspecific serine/threonine-kinase activities were measured both in the absence of phosphatidyl serine and in the presence of 2 mmol/liter EGTA in component buffer. The enzyme activity in the membrane fraction specific for conventional PKC was quantitated by subtracting the nonspecific serine/threonine-kinase activities from total kinase activity.
Northern blot analysis
The cDNA probes used were a 0.8 kb EcoRI fragment encoding human 1(IV) collagen and a 0.6 kb SmaI fragment encoding human TGF-. Each mRNA was isolated from human mesangial cells using QuickPrep (Pharmacia, Uppsala, Sweden). For Northern blot analysis, 0.4 g of mRNA from each sample of human mesangial cells was electrophoresed in 1% agarose gel and transferred to nitrocellulose filter (Nitroplus 2000; Micro Separations, Westboro, MA, USA) for 18 hours at room temperature using 10 SSC as the transfer solution and cross-linked by UV (FS 1500, Funakoshi, Japan) at 12 J/cm2. Prehybridization with salmon sperm DNA was performed for three hours followed by hybridization for eight hours using 50% formamide with a32P-radiolabeled probes (Multiprine; Amersham, Arlington Height, IL, USA). Hybridized filters were washed with 1 SSC (0.1% SDS) for 15 minutes, and twice with 0.1 SSC (0.1% SDS) for 15 minutes at 60°C. The filters were then exposed to Kodak XAR-5 film using an intensifying screen for two days at -80°C.
Statistical analysis
Data were expressed as mean 
SD. Differences between groups were evaluated by paired Student's t-test. A P value less than 5% denoted the presence of a statistically significant difference.
RESULTS
Effect of 5-HT on production of type IV collagen by human mesangial cells
We first examined the in vitro effect of 5-HT on the production of type IV collagen by human mesangial cells. When mesangial cells were incubated with the medium alone for 24 hours, the baseline concentration of type IV collagen was 15 g/mg cell protein, indicating that type IV collagen was constitutively produced by human mesangial cells Figure 1a. Incubation of mesangial cells with various concentrations of 5-HT for 24 hours significantly increased the production of type IV collagen in a dose-dependent manner, and the peak concentration was observed at 10-6 mol/liter of 5-HT Figure 1a. We also examined the kinetics of type IV collagen secretion in the presence or absence of 5-HT. In the control media, the concentration of type IV collagen increased in a time-dependent manner, but its production by mesangial cells was significantly higher, relative to controls, at each time interval when these cells were incubated in the presence of 10-6 mol/liter of 5-HT Figure 1b. When human mesangial cells were incubated with 10-6 mol/liter of 5-HT together with various concentrations of ketanserin or sarpogrelate hydrochloride, 5-HT2A receptor antagonists, 5-HT-induced increase in type IV collagen secretion was completely suppressed by these agents Figure 1c. In contrast, the 5-HT3 receptor antagonist, ondansetron, did not affect 5-HT-induced increase in type IV collagen production Figure 1c. These results suggested that the 5-HT-enhanced production of type IV collagen might be mediated through the 5-HT2A receptor.
Figure 1.
Effects of 5-hydroxytryptamine (5-HT) and 5-HT receptor antagonists on production of type IV collagen by human mesangial cells. (A) Human mesangial cells (5 104 cells/well) were incubated with the indicated concentrations of 5-HT. After 24 hours incubation, type IV collagen concentrations in the medium were determined. Data represent the mean of three separate experiments. Error bars indicate SDs.*P < 0.05, compared with control. (B) Human mesangial cells (5 104 cells/well) were incubated in the absence (
) or the presence (
) of 10-6 mol/liter of 5-HT for 24, 48, and 72 hours. After incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments. (C) Human mesangial cells (5 104 cells/well) were incubated with 10-6 mol/liter of 5-HT in the absence or presence of indicated concentrations of ketanserin (
), ondansetron (
), and sarpogrelate hydrochloride (
). As a control, human mesangial cells were incubated with medium alone (). After 24 hours incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments. *P < 0.05, compared with cells incubated with 10-6mol/liter of 5-HT alone.
Northern blot analysis of type IV collagen 1 showed that 5-HT increased the mRNA level of type IV collagen 1 chain in a time-dependent manner and a significant difference was observed at 24 hours following the addition of 10-6 mol/liter of 5-HT Figure 2. Figure 3 shows that both constitutive and 5-HT-induced type IV collagen production were completely inhibited by cycloheximide Figure 3. These results suggested that 5-HT-enhanced production of type IV collagen is regulated at a transcriptional level.
Figure 2.
Effect of 5-HT on mRNA of type IV collagen 
1 chain by human mesangial cells. Human mesangial cells were incubated with 10-6 mol/liter of 5-HT for indicated time. The mRNA was isolated and 0.4 g of mRNA were separated by electrophoresis in formaldehyde-containing gel and transferred to a nitrocellulose filter. The filter was hybridized with a heat-denatured32P-labeled 1(IV) probe. As a control, 18S and 28S were visualized on a UV monitor.
Figure 3.
Effect of cycloheximide on production of type IV collagen by human mesangial cells. Human mesangial cells (5 104 cells/well) were incubated with () or without () 10-6 mol/liter of 5-HT in the absence or the presence of indicated concentrations of cycloheximide. After 24 hours of incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments. < 0.05, compared with cells incubated with 10-6mol/liter of 5-HT without cycloheximide.*P
Involvement of TGF- in 5-HT-induced increase in type IV collagen production
To elucidate the mechanism of 5-HT-enhanced production of type IV collagen, we examined the involvement of TGF- in this process in the next series of experiments. Bioactivities of both active and total TGF- significantly increased following the addition of 5-HT in a dose-dependent manner, and the peak activity of active and total TGF- occurred in the presence of 10-6 mol/liter of 5-HT Figure 4a. Interestingly, 5-HT-stimulated bioactivity of active TGF- Figure 4a paralleled the rise in type IV collagen secretion Figure 1a. The time course study of TGF- bioactivity showed that both active and total TGF- activities became significant at 12 hours after the addition of 5-HT Figure 4b. Figure 4c shows that the level of TGF- mRNA was increased by 5-HT. These results demonstrated that 5-HT increased both total and active TGF- bioactivities. Figure 5a shows that non-immune IgG failed to change the effect of 5-HT on type IV collagen production, whereas an anti-TGF- antibody significantly inhibited 5-HT-induced type IV collagen production. Moreover, anti-TGF- antibody partially suppressed the constitutive secretion of type IV collagen. The level of anti-TGF- antibody-induced suppression of type IV collagen production was less than the constitutively produced type IV collagen, suggesting the potential involvement of TGF- in 5-HT-enhanced production of type IV collagen, as well as, at least in part, in constitutive secretion. To test this conclusion, we determined the effect of exogenous active TGF- on the production of type IV collagen in the culture medium. Figure 5b shows that the addition of exogenous active TGF- to these cells markedly increased type IV collagen secretion in a dose-dependent manner (up to 100 pmol/liter). Combined together, these results indicate that TGF- may play an important role in 5-HT-enhanced production of type IV collagen.
Figure 4.
Effects of 5-HT on total and active TGF- bioactivities and TGF- mRNA expression. (A) Human mesangial cells (5 104 cells/well) were incubated with the indicated concentrations of 5-HT. After 24 hours incubation, total (upper panel) and active (lower panel) TGF- bioactivities in the medium were determined. Data represent the mean SD of three separate experiments. *P < 0.05, compared with the control. (B) Human mesangial cells (5 104 cells/well) were incubated in the absence () or the presence () of 10-6 mol/liter of 5-HT for indicated times. Total (upper panel) and active (lower panel) TGF- bioactivities in the medium were determined. Data represent the mean SD of three separate experiments. (C) Northern blot analysis of TGF-. Human mesangial cells were incubated with 10-6 mol/liter of 5-HT. The mRNA of these cells was harvested at 0, 12, 24 hours. The mRNA (0.4 g) was separated by electrophoresis in for aldehyde-containing gel and transferred to a nitrocellulose filter. The filter was hybridized with a heat-denatured32P-labeled TGF- probe. As a control, 18S and 28S were visualized on a UV monitor.
Figure 5.
Inhibitory effect of anti-TGF- antibody and effect of exogenous TGF- on type IV collagen production. (A) Human mesangial cells (5 104 cells/well) were incubated with (,) or without (,) 10-6 mol/liter of 5-HT in the absence or presence of indicated concentrations of anti-TGF- antibody (,) or non-immune IgG (,). After 24 hours incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments.*P < 0.05, compared with non-immune IgG. (B) Human mesangial cells (5 104 cells/well) were incubated with the indicated concentrations of exogenous TGF-. After 24 hours incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments. *P < 0.05, compared with the control.
Involvement of protein kinase C in 5-HT-enhanced production of type IV collagen
Figure 6a shows the time course study of membrane PKC activity. The addition of 10-6 mol/liter of 5-HT to human mesangial cells resulted in 2.5-fold rise in enzyme activity (relative to basal level) at five minutes and in 7.0-fold increase above the basal level at 10 minutes. However, this was followed by a rapid decline to levels close to the baseline at 20 minutes Figure 6a. To elucidate the role of PKC on 5-HT-enhanced production of type IV collagen, we examined the effect of a PKC inhibitor, calphostin C, on this process. Incubation of human mesangial cells with 10-6 mol/liter of 5-HT together with various concentrations of calphostin C suppressed 5-HT-induced increase in type IV collagen secretion in a dose-dependent manner, with 50% inhibition at 50 nmol/liter of calphostin C and a maximal inhibition at 100 nmol/liter Figure 6b. Interestingly, calphostin C did not affect the constitutive production of type IV collagen in human mesangial cells Figure 6b. Under the experimental conditions described above, calphostin C was not cytotoxic to human mesangial cells. This conclusion was based on results of the cell counting assay using trypan blue staining and the release of lactic dehydrogenase from these cells (data not shown). These results suggest that PKC may be involved in the production of type IV collagen. To confirm the involvement of PKC in type IV collagen production, we examined the effect of phorbol ester, a well known PKC activator, on type IV collagen production and TGF- activity. Figure 7a shows that phorbol ester induced an increase in type IV collagen production as well as activation of active and total TGF- Figure 7b. These results suggested that 5-HT-induced type IV collagen production was mediated by activation of PKC, which in turn increased TGF- activity in human mesangial cells. To test this notion, we examined the effect of calphostin C on 5-HT-stimulated TGF- bioactivity. A complete inhibition of 5-HT-induced increase in active and total TGF- bioactivities was noted following the addition of 100 nmol/liter of calphostin C Figure 8. Moreover, the effect of exogenous TGF- on the concentration of type IV collagen was not inhibited by calphostin C (data not shown). Thus, our results suggest that 5-HT-induced production of type IV collagen by human mesangial cells is mediated by activation of PKC and the subsequent increase in active TGF- activity.
Figure 6.
Involvement of protein kinase C (PKC) in 5-HT-induced production of type IV collagen. (A) Human mesangial cells (1 107 cells) were incubated with 10-6 mol/liter of 5-HT for the indicated time interval. The activity of membrane PKC was determined. Data represent the mean SD of three separate experiments.*P < 0.05, compared with baseline (0 min). (B) Human mesangial cells (5 104 cells/well) were incubated with () or without () 10-6 mol/liter of 5-HT in the absence or presence of the indicated concentrations of calphostin C. As control, the cells were incubated with 0.1% DMSO in the presence (), or absence () of 10-6 mol/liter 5-HT. After 24 hours of incubation, the concentration of type IV collagen in the medium was determined. Data represent the mean SD of three separate experiments. *P < 0.05, compared with cells incubated with 10-6 mol/liter of 5-HT without calphostin C.
Figure 7.
Effect of phorbol ester on production of type IV collagen and TGF- activity. (A) Human mesangial cells (5 104 cells/well) were incubated with or without 10 nmol/liter of phorbol 12-myristate 13-acetate (TPA). After 24 hours incubation, the concentration of type IV collagen in the medium was determined. (B) Human mesangial cells (5 104 cells/well) were incubated with 10 nmol/liter of TPA. After 24 hours of incubation, total (upper panel) and active (lower panel) TGF- bioactivities in the medium were determined. Data represent the mean SD of three separate experiments.
Figure 8.
Inhibitory effect of calphostin C on 5-HT-enhanced bioactivities of total and active TGF- in human mesangial cells. Human mesangial cells (5 104 cells/well) were incubated with () or without () 10-6 mol/liter of 5-HT in the absence or presence of the indicated concentrations of calphostin C. After 24 hours of incubation, total (upper panel) and active (lower panel) TGF- bioactivities in the medium were determined. Data represent the mean SD of three separate experiments. *P < 0.05, compared with cells incubated with 10-6 mol/liter of 5-HT without calphostin C.
DISCUSSION
The major findings of the present study were that 5-HT enhanced the production of type IV collagen by human mesangial cells in the kidney, and that this effect was mediated by activation of PKC with a subsequent increase in active TGF- bioactivity. These conclusions were supported by the following findings: (1) production of type IV collagen by human mesangial cells increased following the addition of 5-HT to the medium Figure 1 a, b, which was inhibited by 5-HT2A receptor antagonists Figure 1c; (2) the bioactivities of both active and total TGF- were increased by 5-HT Figure 4; (3) 5-HT-enhanced production of type IV collagen was completely inhibited by an anti-TGF- antibody Figure 5a; (4) the activity of membrane PKC was increased by 5-HT Figure 6a; (5) inhibition of PKC by calphostin C suppressed the stimulatory effect of 5-HT on type IV collagen production Figure 6b; (6) phorbol ester enhanced activation of active TGF- and production of type IV collagen Figure 7; (7) increased bioactivity of TGF- by 5-HT was completely inhibited by calphostin C Figure 8; and (8) the exogenous TGF--induced increase in type IV collagen secretion Figure 5b was not inhibited by calphostin C (data not shown).
The 5-HT2A subreceptor type is present in high concentrations in the brain, platelets, vascular smooth muscle cells and glomerular mesangial cells3,6,20,21,22,23. Ketanserin is a well known 5-HT2A receptor antagonist3,24. Sarpogrelate hydrochloride is also a selective 5-HT2A receptor antagonist that inhibits 5-HT-induced platelet aggregation25 and constriction of vascular smooth muscle cells25. In the present study, the 5-HT-induced increase in type IV collagen secretion by human mesangial cells was completely suppressed by these 5-HT2A receptor antagonists but not by the 5-HT3 receptor antagonist, ondansetron Figure 1c26, suggesting that the effect of 5-HT was, at least in part, mediated through 5-HT2A subreceptor type present on human mesangial cells.
Our results also showed that calphostin C inhibited the stimulatory effect of 5-HT on the secretion of type IV collagen Figure 6b, while the activity of membrane PKC was increased by 5-HT Figure 6a. Moreover, phorbol ester, a well known PKC activator, increased the production of type IV collagen Figure 7a. These results strongly suggest that the effect of 5-HT on type IV collagen secretion is mediated by activation of PKC in mesangial cells. PKC is involved in agonist-induced cellular responses in various cell types27,28,29. The enzyme is activated by high concentrations of diacylglycerol (DAG) and calcium in the cell membrane, which are derived from agonist-induced hydrolysis of inositol phospholipid27. In cultured rat glomerular mesangial cells, 5-HT stimulates the proliferation of these cells and prostaglandin synthesis by the same cells16,30, which are mediated by stimulation of phospholipase C16,30. This pathway of cellular signals induces a transient rise in cytosolic calcium followed by activation of PKC16. Thus, it is possible that 5-HT-enhanced production of type IV collagen by human mesangial cells is mediated by activation of phospholipase C, and subsequently by a rise in DAG and calcium, and then further by activation of PKC. Recently, Koya et al reported that PKC beta was activated in the glomeruli of diabetic rats31. Moreover, an oral PKC beta inhibitor ameliorated renal dysfunction of diabetic rats32. These studies suggested that PKC beta might play an important role in the development of diabetic nephropathy. However, the PKC family consists of at least 11 different subspecies of serine/threonine protein kinases33, such as conventional PKC (, 1, 2 and 
) and novel PKC (
, 
, 
and 
). Recent studies have also reported the presence of atypical subspecies of PKC33. Therefore, further studies are necessary to determine the exact PKC isozyme(s) involved in 5-HT-enhanced production of type IV collagen by human mesangial cells.
5-HT enhanced the production of type IV collagen from mesangial cells Figure 1 a, b and increased the activity of active TGF- Figure 4. Moreover, an anti-TGF- antibody induced a complete suppression of type IV collagen production Figure 5a. Furthermore, exogenous active TGF- enhanced the type IV collagen production Figure 5b. These results suggest the possible involvement of TGF- in 5-HT-enhanced production of type IV collagen by mesangial cells. Does the effect of 5-HT on the activity of active TGF- represent a direct effect or indirect effect through activation of PKC? Our results showed that calphostin C inhibited the effect of 5-HT on collagen secretion Figure 6b, suppressed the effects of 5-HT on TGF- Figure 8, but did not affect exogenous active TGF--induced type IV collagen production (data not shown). Moreover, the time course studies demonstrated that PKC activity showed peak activity at 10 minutes Figure 6a, TGF- activity become significant at 12 hours Figure 4b, and collagen production become significant at 24 hours following the addition of 5-HT Figure 1b. These results suggest that the effect of 5-HT on TGF- are probably mediated by activation of PKC. This conclusion is further supported by results of previous studies from other laboratories. For example, Akhurst, Fee and Balmain reported that phorbol esters stimulated the expression of TGF- mRNA in mouse epidermal cells34. Moreover, angiotensin II stimulated the induction of TGF- mRNA via PKC activation in rat smooth muscle cells35. Furthermore, phorbol esters induced both PKC activation and TGF- mRNA in rat mesangial cells36. In mesangial cells, several activators of PKC have been shown to increase TGF- mRNA and bioactivity. These activators include high concentrations of glucose in mice37, angiotensin II in rats38, thromboxane in rats39, and low density lipoproteins in rats40, all of which also increase mesangial cell matrix protein synthesis by mesangial cells.
TGF- is usually secreted as a biologically inactive form, latent TGF-41. There are three components of the latent TGF- complex: (1) active TGF-, (2) TGF- latency-associated peptide (LAP), and (3) latent TGF- binding protein (LTAP)41. Since TGF- is secreted in a latent form, the dissociation of active TGF- from the latent complex is essential for the expression of its biological activities post secretion. Latent TGF- is activated by proteolysis of the latent complex with plasmin and cathepsin D, or by alteration of carbohydrate structures within LAP. Plasmin cleaves LAP and releases active TGF- from latent TGF-. Tissue-type plasminogen activator (tPA) converts plasminogen to the proteolytic active plasmin. Activation of PKC has been shown to induce tPA in HeLa, HepG2, and Bowes melanoma cells42. In fact, human mesangial cells also secrete tPA43. Accordingly, it is possible that 5-HT-stimulated PKC activation increases tPA activity, thereby enhancing the activation of latent TGF- via an increased level of plasmin.
The signal pathway downstream TGF- activation to the secretion of type IV collagen in human mesangial cells remains unknown. However, previous studies using cultured mouse or rat mesangial cells showed that exogenous TGF- increases the production of collagen, fibronectin and proteoglycan on a protein level11,44. TGF- also increases mRNA expression of types I and IV collagen in rat mesangial cells12. Moreover, TGF- is reported to promote the deposition of extracellular matrix by stimulating the synthesis of matrix proteins, increase the activity of tissue inhibitor of matrix-degrading metalloproteinases, and/or inhibit the expression of stromelysin, which are enzymes involved in the metabolism of extracellular matrix45,46.
In the present study, 5-HT-enhanced production of type IV collagen was observed at concentrations from 10-7 mol/liter Figure 1a. Interestingly, the concentrations of plasma 5-HT in diabetic patients ranged from 10-8 to 10-7 mol/liter4,5. Although the exact concentration and sources of 5-HT in mesangial lesions in diabetic patients are still unknown, it is possible that the high plasma concentrations of 5-HT in diabetic patients may enhance the production of type IV collagen by glomerular mesangial cells, thereby enhancing the expansion of mesangial lesions. Further studies are needed in order to elucidate this issue.
In conclusion, the present in vitro experiments demonstrated that 5-HT enhanced the production of type IV collagen by human mesangial cells, which was mediated by activation of PKC and subsequent increase in the activity of active TGF-.
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Acknowledgments
We are grateful to Drs. Toshihiro Yano and Takayuki Higashi for their collaborative endeavor, and to Mr. Kenshi Ichinose for his technical assistance. We are also grateful to Dr. Tetsuto Kanzaki at the 2nd Dept. of Internal Medicine, School of Medicine, Chiba University, for providing TGF- cDNA, and to Dr. Shogo Nakano at the Dept. of Surgical Pathology, Kumamoto University School of Medicine, and Dr. Hidekatsu Yoshioka at the 2nd Dept. of Biochemistry, Oita Medical University, for providing collagen type IV 1 cDNA.
