Background

Our previous studies have demonstrated that renal proximal tubular epithelial cells (PTCs) may contribute to renal interstitial fibrosis by the generation of transforming growth factor-1 (TGF-1). In these in vitro experiments, TGF-1 was, however, secreted in its latent form. Plasmin has been implicated as a potential physiological activator of TGF-1. The inter--trypsin inhibitor (II) family of serum protease inhibitors together with tumor necrosis factor-stimulated gene 6 (TSG-6) recently have been implicated in the regulation of this protease pathway. The aim of the current study was to examine PTC synthesis of these proteins and to relate it to alterations of plasmin-protease activity.

Methods

PTCs were grown to confluence and stimulated under serum-free conditions with either interleukin-1 (IL-1) or 25 mmol/L D-glucose. Alterations in II and TSG-6 generation were detected by Western analysis of both membrane extracts and supernatant samples. Alterations in gene expression were examined by reverse transcription-polymerase chain reaction. The effect of alteration in synthesis of TSG-6 on plasmin activity was determined by quantitating plasmin inhibitory activity of supernatant samples by in vitro calorimetric assay prior to and following TSG-6 immunoprecipitation.

Results

The data demonstrate that human PTCs constitutively express mRNA for bikunin and heavy chain 3 (H3) of II. Neither IL-1 (1 ng/mL) nor 25 mmol/L D-glucose influenced their mRNA expression nor protein synthesis. In contrast, the addition of either IL-1 or 25 mmol/L D-glucose increased TSG-6 mRNA expression. This was accompanied by an early up-regulation of TSG-6 protein expression following IL-1 stimulation (24 h) and a late up-regulation after the addition of 25 mmol/L D-glucose (96 h) in the cell culture supernatant and associated with the cell membranes. Early induction of TSG-6 mRNA by IL-1 was unaffected by the addition of the protein synthesis inhibitor cycloheximide. In contrast, the later glucose-stimulated induction of TSG-6 mRNA was abrogated by the addition of cycloheximide. Stimulation of TSG-6 by either IL-1 or 25 mmol/L D-glucose was associated with an inhibition of total percentage plasmin activity. Immunoprecipitation of TSG-6 in these samples returned plasmin activity to control levels.

Conclusions

The data demonstrate that human PTCs constitutively express the bikunin and H3 components of the II family of serum protease inhibitors. Moreover, the addition of IL-1 or 25 mmol/L D-glucose up-regulates the expression of TSG-6 in these cells, resulting in an inhibition of plasmin activity.

METHODS

Cell culture

HK2 cells [human renal PTCs immortalized by transduction with human papilloma virus (HPV) 16 E6/E7 genes]¹⁰ were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (FCS; Biological Industries Ltd., Cumbernauld, UK), glutamine (Life Technologies Ltd.), HEPES buffer (GIBCO BRL, Paisley, UK), insulin, transferrin, and sodium selenite (Sigma, Poole, UK). Cells were grown at 37°C in 5% CO₂ and 95% air. Fresh growth medium was added to cells every three to four days until confluent. Cells were grown to confluence and serum deprived for 72 hours prior to experimental manipulation. All experiments were performed under serum-free conditions.

Selective experiments with primary HPTCs were performed to confirm key observations obtained in HK2 cells. HPTCs were isolated from normal tissue obtained from nephrectomy specimens, characterized as previously described¹ and grown under the same culture conditions as HK2 cells.

Examination of II and TSG-6 gene expression

Confluent growth-arrested monolayers of HK2 cells or HPTCs were stimulated with either IL-1 (1 ng/mL) or 25 mmol/L D-glucose in the absence of serum. In control experiments, cells were exposed to either 5 mmol/L D-glucose or 25 mmol/L L-glucose as the "normal glucose" and "hyperosmolar" controls, respectively. Total cellular RNA was extracted, and gene expression of components of the II family of proteins and TSG-6 was examined at various time points up to 96-hours poststimulation by reverse transcription (RT) and polymerase chain reaction (PCR) amplification as previously described¹. PCR amplification was performed over a range of cycle numbers (26 to 38) to ensure that the amplification was in the linear range of the curve. One tenth of the PCR reaction from both test and control (-actin) product was mixed and separated by flat-bed electrophoresis in 3% wt/vol NuSieve GTG agarose gels (Flowgen Instruments Ltd., Sittingbourne, UK), stained with ethidium bromide (Sigma), and photographed. The negatives were scanned using a densitometer (Model 620 video densitometer; Bio-Rad Laboratories Ltd., Hemelhempstead, UK) and the density of the bands compared with those of the housekeeping gene.

The role of protein synthesis in either IL-1 or 25 mmol/L D-glucose induction of TSG-6 mRNA was determined by stimulation of confluent growth-arrested monolayers of HK2 cells with either IL-1 (1 ng/mL) or 25 mmol/L D-glucose in the presence of cycloheximide (5 g/mL). Our previous studies have demonstrated that at this dose, cycloheximide inhibits protein synthesis in the absence of cell toxicity, as assessed by quantitation of total cellular adenosine 5'-triphosphate (ATP)¹¹. Total cellular RNA was extracted, and gene expression of TSG-6 mRNA was examined by RT-PCR using specific oligonucleotide primers at time points of up to 72-hours poststimulation as described previously in this article Table 1.

Table 1 - The sequences of the polymerase chain reaction (PCR) amplification primers.

Full table

Generation and purification of TSG-6 antibody

Antibodies against a synthetic peptide of TSG-6 (CGGVFTDPKRIFKSPG, residue 136 to 150) were raised in rabbits. The antiserum was subsequently purified using Sulfolink-coupling gel (Pierce, Rockford, IL, USA). Briefly, 5 mL of Sulfolink resin were packed in a disposable column (Bio-Rad), and the column was equilibrated with 30 mL coupling buffer [50 mmol/L Tris/HCl, 5 mmol/L Na₂ ethylenediaminetetraacetic acid (EDTA), pH 8.5]. Five milligrams of the synthetic TSG-6 peptide (Cys136-Gly150) were dissolved in coupling buffer and the resin overlaid with the peptide solution. The column was covered with aluminum foil to keep out light, sealed and then rotated end to end for 15 minutes. After standing vertically for 30 minutes in the dark, the excess coupling solution was drained, and the resin was washed with a further 15 mL of coupling buffer. Uncoupled sites on the resin were then blocked using cysteine (50 mmol/L in coupling buffer). The resin was again washed with 15 mL coupling buffer, followed by 80 mL 1% acetic acid and 80 mL NaCl. Finally, the peptide (Cys136-Gly150)-affinity resin was washed with 50 mL 0.05% NaN₃ and stored in the dark at 4°C. Prior to purification of the antipeptide (Cys136-Gly150) antibodies, the column was washed with 40 mL phosphate-buffered saline (PBS). The antiserum was then pumped through the column at room temperature at 1 mL/h. The resin was washed with 80 mL PBS, followed by 80 mL 1 mol/L NaCl in phosphate buffer, and finally 40 mL PBS. The bound antibodies were eluted with 0.1 mol/L glycine (pH 2.8), and the eluate was collected as 2 mL fractions. The fractions were immediately neutralized by adding 100 L 1 mol/L Tris/HCl (pH 9.5), and the optical density (OD) of the neutralized fractions was read at 280 nm. The fractions containing protein were pooled and extensively dialyzed against PBS at 4°C. Finally, the OD was read at 280 nm to calculate the protein content, and aliquots were stored at -70°C. The specificity of the antibody was confirmed by its ability to detect recombinant TSG-6 (gift from Dr. Wisniewski, New York, NY, USA) and TSG-6 in whole serum and synovial fluid by Western analysis (data not shown).

Examination of II and TSG-6 protein expression

Confluent growth-arrested monolayers of HK2 cells were stimulated with either IL-1 (1 ng/mL) or 25 mmol/L D-glucose in the absence of serum. To ensure that II/bikunin was not derived from serum, cells were grown in 10% FCS from which glycosaminoglycans (and therefore bikunin) were removed as previously described¹², following seeding until growth arrest. Briefly, FCS was passed over DEAE Sephacel (Pharmacia, Uppsala, Sweden) to remove any exogenous bovine glycosaminoglycans/proteoglycans. The removal of II was confirmed by Western analysis of the deglycosaminoglycated FCS (data not shown). In control experiments, cells were exposed to either 5 mmol/L D-glucose or 25 mmol/L L-glucose.

Following stimulation of cells, supernatants were collected and concentrated eightfold using centricon microconcentrators (YM-10; 10,000 molecular weight cutoff; Amicon, Stonehouse, UK). In parallel experiments following removal of supernatant, the cell surface expression of II and TSG-6 was examined by Western analysis of cell membrane isolates. Preparations of HK2 cell membranes were performed as previously described¹³. Briefly, cells were washed three times in PBS and scraped off the flasks into 6 mL PBS containing a cocktail of proteinase inhibitors [pepstatin A, aprotinin, and leupeptin at 10 g/mL, and phenylmethylsulfonyl fluoride (PMSF) at 174 g/mL; all purchased from Sigma]. Cells were pelleted by centrifuging at 500 g for 10 minutes at 4°C. The cell pellet was subsequently resuspended in 0.4 mL hypotonic buffer (10 mmol/L Tris, 1 mmol/L MgCl₂, pH 7.2, containing the protease inhibitor cocktail) and mixed with four volumes of sucrose buffer (0.25 mol/L sucrose dissolved in hypotonic buffer). Membranes were homogenized by 50 strokes in a dounce pestle and centrifuged at 500 g for five minutes at 4°C to remove nuclei and unbroken cells. A membrane-enriched fraction was obtained by centrifuging the supernatant at 25,000 g for 30 minutes at 4°C. The resulting membrane pellets were resuspended in PBS containing 1 mmol/L PMSF. The protein content of the samples was determined using the Bio-Rad protein assay (Bio-Rad Laboratories GmbH, München, Germany) using bovine serum albumin (Sigma) as a standard.

In all experiments, expression of II and TSG-6 following stimulation was examined by Western analysis. Briefly, equal amounts of cell membrane protein were loaded onto 3 to 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gradient gels and electrophoresis carried out under reducing conditions according to the procedure of Laemmli¹⁴. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, Buckinghamshire, UK). The membrane was blocked with Tris-buffered saline containing 5% nonfat powdered milk for one hour and then incubated with either rabbit anti-human II (Dako, Glostrup, Denmark) or purified polyclonal rabbit anti–TSG-6 antibody raised against a synthetic peptide of TSG-6 (discussed previously in this article) in Tris-buffered saline containing 1% bovine serum albumin and 0.05% Tween 80 (Tris-buffered saline-Tween) for one hour at room temperature. The blots were subsequently washed in Tris-buffered saline-Tween and then incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma) in Tris-buffered saline-Tween. Proteins were visualized using enhanced chemiluminescence (Amersham) according to the manufacturer's instructions. The specificity of the antihuman II has been previously demonstrated in human plasma^15,16. The antibody recognizes the heavy chains and bikunin of all II family members, as well as II associated with TSG-6.

Plasmin assay

Plasmin inhibitory activity of supernatant samples was determined by calorimetric assay using H-D-valyl-L-leucyl-L-lysine-p-nitroaniline dihydrochloride (Chromogenix, Milan, Italy) as a chromogenic substrate as previously described⁹. Briefly, nonconcentrated cell supernatants were preincubated with plasmin (Sigma) for 10 minutes at 37°C to allow the formation of plasmin inhibitor complexes. Subsequently, the chromogenic substrate was added, and the reaction mixtures were incubated at 37°C for 10 minutes. The reaction was stopped by the addition of acetic acid, and the degree of proteolysis was determined by measuring adsorbance at 405 nm. The final concentrations in the assay were 0.7 mmol/L H-D-valyl-L-leucyl-L-lysine-p-nitroaniline dihydrochloride and 0.07 CU/mL plasmin. Each supernatant sample was measured in duplicate, and the OD values obtained normalized to the time zero medium control for each experimental condition.

Immunoprecipitation of TSG-6

Confirmation that alterations in TSG-6 were responsible for changes in plasmin inhibitory activity was sought by repeating plasmin assays following TSG-6 immunoprecipitation. Briefly, cell supernatants were precleared with 50 L packed protein A cross-linked 4% beaded agarose (Sigma) at 4°C for one hour. The beads were removed by centrifugation (13,000 r.p.m., 5 sec), and the supernatant was collected; 10.5 g of purified polyclonal rabbit anti–TSG-6 antibody (discussed previously in this article) or purified rabbit IgG (Sigma) as a control was added to each 500 L of the cleared supernatant and incubated at 4°C with constant mixing for 12 hours. The immune complex was captured by the addition of packed agarose protein A beads (100 L/500 L supernatant) for two hours at 4°C. Separation of the beads was achieved by centrifugation (13,000 g for 5 min), and the supernatant was removed. Plasmin assay of the cell supernatants following immunoprecipitation was then performed, and results were compared with supernatant aliquots from the same experiments that had not undergone TSG-6 immunoprecipitation.

Statistical analysis

Values are expressed as mean SD. Values are compared using the Student t test. Statistical significance was defined as P < 0.05.

RESULTS

Regulation of II: Constitutive expression of bikunin and H3

Confluent monolayers of growth-arrested HK2 cells demonstrated constitutive expressions of bikunin and H3 mRNA of II. The addition of either IL-1 or 25 mmol/L D-glucose to confluent monolayers of HK2 cells did not influence bikunin or H3 gene expression Figure 2. Similarly, the addition of either IL-1 or 25 mmol/L D-glucose to confluent monolayers of HPTCs did not stimulate expression of the mRNA of II components (bikunin and H3 chains; data not shown). No expression of H1 chain mRNA of II was detected in either stimulated or unstimulated HK2 or HPTCs by RT-PCR with an amplification up to 42 cycles (liver homogenates served as positive controls).

Figure 2.

Constitutive expression of bikunin and H3 mRNA. Growth-arrested HK2 cells were stimulated with 5 mmol/L D-glucose, 25 mmol/L D-glucose, or interleukin (IL)-1 (1 ng/mL) in the absence of serum. Total cellular RNA was isolated and mRNAs for bikunin (A) and heavy chain 3 (H3) mRNA (B) examined by RT-PCR (36 cycles of amplification). Ethidium bromide stained PCR products were separated on a 3% agarose gel. Scanning densitometry confirmed the lack of induction of either bikunin or H3 mRNA expression in the presence of either IL-1 or 25 mmol/L D-glucose. One representative gel of four individual experiments is shown.

Full figure and legend (68K)

Western blot analysis confirmed the constitutive expression of proteins of the II family in the membrane extracts of HK2 cells. None of the components were influenced by addition of either IL-1 or 25 mmol/L D-glucose Figure 3.

Figure 3.

Protein expression of the inter-alpha-trypsin inhibitor (II) family in membrane isolates of HK2 cells. Growth-arrested HK2 cells were stimulated with either 5 mmol/L D-glucose (5 Gl) or IL-1 (1 ng/mL) for up to 24 hours in the absence of serum. Membrane preparations were isolated, and equal amounts of HK2-derived membrane protein (19 g/lane) were loaded onto 3 to 12% SDS-PAGE gradient gels. Western blot analysis was carried out as detailed in the Methods section. In parallel experiments, growth-arrested cells were stimulated with either 5 mmol/L D-glucose (5 Gl), 25 mmol/L D-glucose (25 Gl), or 25 mmol/L L-glucose (25 L-Gl), for up to 96 hours. The antiserum used recognizes intact II family members [including pre-I (120 to 130 kD)], individual heavy chains (75 to 80 kD), and bikunin (35 kD), as well as II associated with TSG-6 (TSG-6, molecular weight 42 kD). The multiple bands following Western analysis therefore represent intact II family members, individual chains of II, and complexes bound to TSG-6¹⁸. Scanning densitometry confirmed that there were no differences in II expression under any of the experimental conditions. One representative experiment of four independent experiments is shown.

Full figure and legend (107K)

Induction of TSG-6 mRNA and protein by IL-1 and glucose

Stimulation of growth-arrested monolayers of HK2 cells with either IL-1 or 25 mmol/L D-glucose led to induction of TSG-6 mRNA. This was apparent 3 hours after the addition of IL-1, but only 48 hours after the addition of 25 mmol/L D-glucose Figure 4a. Induction of TSG-6 mRNA was also detected following stimulation of HPTCs by either IL-1 or 25 mmol/L D-glucose Figure 4b. Increased expression of TSG-6 mRNA in HPTCs showed similar kinetics to that seen in HK-2 cells, with delayed up-regulation in the presence of 25 mmol/L D-glucose relative to that seen after IL-1 stimulation.

Figure 4.

Induction of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA by interleukin-1 (IL-1) or 25 mmol/L D-glucose. Growth-arrested HK2 cells (A) or HPTCs (B) were stimulated with 5 mmol/L D-glucose, 25 mmol/L D-glucose, or IL-1 (1 ng/mL) in the absence of serum. Total cellular RNA was isolated at various time points up to 96 hours, and TSG-6 mRNA was examined by RT-PCR. Ethidium bromide-stained PCR products were separated on a 3% agarose gel. One representative gel of four individual experiments is shown.

Full figure and legend (47K)

The induction of TSG-6 mRNA by either stimulation by IL-1 or addition of 25 mmol/L D-glucose was accompanied by an increase in TSG-6 protein expression in the membrane extracts and supernatants of HK2 cells, as detected by Western analysis Figures 5 and 6. Twenty-five mmol/L L-glucose, serving as an osmotic control, did not induce TSG-6 mRNA (data not shown) nor protein expression Figure 6.

Figure 5.

Induction of TSG-6 protein following IL-1 stimulation. Growth-arrested HK2 cells were stimulated with 5 mmol/L D-glucose (5 Gl) or IL-1 (1 ng/mL) in the absence of serum. Membrane preparations were isolated at 24-hours poststimulation (A). Equal amounts of PTC-derived membrane protein (18 g/lane) was loaded onto 3 to 12% SDS-PAGE gradient gels. In parallel experiments, supernatants were collected, concentrated, and loaded onto 3 to 12% SDS polyacrylamide gradient gels (20 L of 8, concentrated supernatant/lane; B). Western blot analysis was carried out as detailed in the Methods section.

Full figure and legend (76K)

Figure 6.

Induction of TSG-6 protein following 25 mmol/L D-glucose stimulation. Growth-arrested HK2 cells were stimulated with 5 mmol/L D-glucose (5 Gl) or 25 mmol/L D-glucose (25 Gl) or 25 mmol/L L-glucose (25 L-Gl). Membrane preparations (A) or supernatant samples (B) were collected at 72-hours and 96-hours poststimulation, respectively. Equal amounts of PTC-derived membrane protein (19 g/lane) were loaded onto 3 to 12% SDS-PAGE gradient gels. Supernatant were concentrated using centricon microconcentrators and loaded onto 3 to 12% SDS-PAGE gradient gels (20 L/lane). Western blot analysis was carried out as detailed in the Methods section.

Full figure and legend (114K)

Induction of TSG-6 mRNA by IL-1 is regulated via a protein synthesis-independent pathway

To assess whether the IL-1 and 25 mmol/L D-glucose–induced TSG-6 mRNA expression was dependent on de novo protein synthesis, HK2 cells were stimulated with either 1 ng/mL of IL-1 or 25 mmol/L D-glucose in the presence of cycloheximide (5 g/mL). The addition of cycloheximide alone did not influence TSG-6 mRNA expression at any of the time points studied Figure 7. While IL-1–induced TSG-6 mRNA up-regulation was not influenced by cycloheximide, the effect of 25 mmol/L D-glucose was abrogated by cycloheximide Figure 7. This observation suggests that 25 mmol/L D-glucose can exert a stimulatory effect on TSG-6 synthesis via de novo protein synthesis.

Figure 7.

Effect of cycloheximide upon the IL-1 and 25 mmol/L D-glucose–induced TSG-6 mRNA. Growth-arrested HK-2 cells were stimulated with IL-1 (1 ng/mL) in the presence or absence of cycloheximide (5 g/mL) for up to 24 hours. Similarly, growth-arrested cells were stimulated with 25 mmol/L D-glucose in the presence or absence of cycloheximide (5 g/mL) for up to 72 hours prior to isolation of total cellular RNA. In addition, RNA was isolated from unstimulated control cells at time zero, and cells were incubated with cycloheximide alone at time points up to 72 hours. TSG-6 mRNA was examined by RT-PCR. Subsequently, PCR products were separated by electrophoresis on a 3% agarose gel (A). The results are expressed as the normalized densitometric ratios of TSG-6 mRNA to -actin following stimulation with either 25 mmol/L D-glucose or IL-1 (1 ng/mL) in the absence of cycloheximide () to the ratio following stimulation in the presence of cycloheximide () (B). One representative gel of four individual experiments is shown.

Full figure and legend (47K)

Inhibition of plasmin activity

Stimulation of HK2 cells by IL-1 resulted in an early increase in plasmin inhibitory activity of cell supernatants Figure 8. The later stimulation of TSG-6 following the addition of 25 mmol/L D-glucose was associated with increased plasmin inhibitory activity at these later time points Figure 8a. A similar pattern of increased in plasmin inhibitory activity was seen following stimulation of HPTCs with IL-1 or 25 mmol/L D-glucose Figure 8b. To confirm that the inhibition of plasmin activity of supernatants from either HK2 cells or HPTCs stimulated with either IL-1 or 25 mmol/L D-glucose was dependent on the stimulation of TSG-6 synthesis, TSG-6 immunoprecipitation was performed and plasmin activity was quantitated. Immunoprecipitation of TSG-6 resulted in removal of all plasmin inhibitory activity from both HK-2 Figure 9a and HPTC culture supernatants. Immunoprecipitation with rabbit IgG had no effect on plasmin inhibitory activity.

Figure 8.

Inhibition of plasmin activity in supernatants after stimulation with IL-1 or 25 mmol/L D-glucose. Growth-arrested HK-2 cells (A) or HPTCs (B) were stimulated with 5 mmol/L D-glucose (), 25 mmol/L D-glucose (), or IL-1 (; 1 ng/mL) in the absence of serum up to 96 hours. Plasmin activity of supernatants was determined as detailed in the Methods section. Results were normalized to the time zero medium control for each experimental condition and the data represents mean SD of three independent experiments. *P < 0.05 IL-1 vs. 5 mmol/L D-glucose; #P < 0.05 25 mmol/L vs. 5 mmol/L D-glucose.

Full figure and legend (29K)

Figure 9.

Immunoprecipitation of TSG-6 abrogates plasmin inhibitory activity in IL-1 and 25 mmol/L D-glucose stimulated cell culture supernatants. Supernatant samples were collected from HK-2 (A) cells or HPTCs (B) stimulated with either IL-1 (1 ng/mL) for 24 hours or 25 mmol/L D-glucose for 96 hours. TSG-6 was immunoprecipitated and plasmin activity measured. Data were normalized to the time zero medium control for each experimental condition. Results show the plasmin activity prior () to and following immunoprecipitation with anti-TSG-6 antibody () or rabbit IgG control (). Values represent the mean SD of four independent experiments. *P < 0.05 for TSG-6 immunoprecipitated samples vs. nonimmunoprecipitated and rabbit IgG control.

Full figure and legend (33K)

DISCUSSION

Previously, we have demonstrated that the co-operative effects of the proinflammatory cytokine IL-1 and elevated D-glucose concentrations stimulate the synthesis of TGF-1, predominately in its latent form². To date, the mode of activation of TGF-1 in vivo remains unclear, although increasing evidence suggests that proteolytic activation by plasmin may represent one physiologically important mechanism¹⁷. A recent study demonstrated that the interactions between II and TSG-6 potentiate the inhibitory effect of II on plasmin activity⁹. We speculate that modification of plasmin activity by II/TSG-6 may subsequently influence the activation of TGF-. As a first step aimed at understanding the mechanisms that underlie the generation of TGF-1 in its latent form, we have examined the regulation of II and TSG-6 generation by renal proximal tubular cells, in response to the stimuli previously implicated in induction of TGF-1 synthesis, namely IL-1 and 25 mmol/L D-glucose.

The data demonstrate that HPTCs constitutively express components of the II family of serum protease inhibitors and that their expression was not influenced by addition of either IL-1 or 25 mmol/L D-glucose. Previous studies have demonstrated that the components of the II family are synthesized predominantly in hepatocytes, and during their transport to the cell surface, the heavy chains become covalently linked to the chondroitin sulfate chain of bikunin. To date, little is known about extra-hepatic synthesis of these components of protease inhibitors. A recent study has demonstrated increased expression of bikunin mRNA transcripts in whole kidney tissue in response to ethylene glycol-induced hyperoxaluria¹⁸. Our studies suggest that renal proximal tubular cells produce the preI variant of II, composed of a single H3 chain associated with bikunin. As we have not excluded the possibility that PTCs may synthesize H2 chains, it remains possible that PTCs may also synthesize ILI. Interestingly, it has been postulated that the II and ILI family members behave as negative acute-phase proteins, while preI is a positive acute phase protein¹⁹, its hepatic synthesis being increased in patients with acute systemic infections.

Inter--trypsin inhibitor family members are captured onto the cell surface through covalent interactions between their heavy chains and hyaluronic acid^20,21. This interaction is subsequently thought to stabilize the pericellular matrix. A unique feature of the inhibition of plasmin activity by II is that in addition to inhibiting soluble plasmin, cell-receptor plasmin activity is also inhibited⁸. This inhibitory effect on receptor/cell surface bound plasmin is in contrast to the more abundant circulating protease inhibitors such as 2-macroglobulin. Recent studies have also demonstrated that plasmin may be the physiological activator of latent TGF-1^17,22,23. Activation of TGF-1 by plasmin is promoted by the surface localization of plasmin by its receptor²⁴ and requires the binding of latent TGF-1 to the plasma membrane^25,26,27. This has led to the proposal of a general model of TGF-1 activation, which involves localization of TGF-1 to the cell surface and its activation by cell-associated plasmin⁶. These observations therefore suggest that modification of cell surface plasmin activity by II may influence the activation of TGF-. In our system, however, it would appear that the synthesis of II components is not influenced by stimuli that induce TGF-1 synthesis.

Tumor necrosis factor-stimulated gene 6 is a recently identified member of the family of the hyaluronan-binding proteins²⁸. Inducible expression of TSG-6 by proinflammatory cytokines has been demonstrated previously in human synoviocytes, and the presence of increased levels of its protein in the synovial fluids of arthritis patients has suggested a role in inflammation²⁹. It is now clear that TSG-6 has potent anti-inflammatory actions both in vitro and in vivo through the formation of a stable complex between TSG-6 and II family members³⁰, which potentiate the plasmin inhibitory activity of the bikunin component of the latter⁹. In contrast to bikunin and H3 chains of II, the data in the current study demonstrate that TSG-6 expression in PTCs was up-regulated upon stimulation with either IL-1 or 25 mmol/L D-glucose. Furthermore, this was associated with reduced plasmin activity. This observation cannot be explained by the induction or inhibition of factors that control the generation of plasmin from plasminogen, since the assay measures the activity of exogenous added plasmin. Furthermore, the immunoprecipitation of TSG-6 from the samples removed all plasmin inhibitory activity, thereby confirming that the inhibition of plasmin activity was dependent on the stimulation of TSG-6. These results suggest that stimuli that we have previously demonstrated to increase the synthesis of TGF-1 also lead to an inhibition of one of the potential pathways leading to its activation.

In other cell systems, IL-1 and glucose have been implicated in the modulation of plasmin activity by modulation of the plasminogen activator (PA)/tissue-type plasminogen activator inhibitor (tPA/urokinase) system. Data obtained in human umbilical vein endothelial cells (HUVECs) suggest that elevated glucose up-regulates t-PA as well as PAI-1 expression and production³¹. This resulted in a decrease in PA activity assessed as the activity of plasmin generated from exogenous added plasminogen. It was concluded that this reflected an overwhelming effect of the increased availability of PAI-1 over t-PA. However, the expression of protease inhibitors, such as the II/TSG-6 system, acting directly on the activity of plasmin downstream from plasminogen was not addressed. Therefore, it cannot be excluded that induction of the II/TSG-6 system, as seen in our study, contributed to the observed net effect on PA activity. In contrast, stimulation of human mesangial cells with IL-1 has been reported to down-regulate PAI-1 production and up-regulate t-PA³². However, the net effect of these changes on plasmin activity was not addressed. Further studies are therefore needed to investigate the upstream events that may regulate plasmin activity in PTCs.

A striking finding of our study was the apparent difference in the kinetics of induction of TSG-6 by IL-1 and 25 mmol/L D-glucose. Furthermore, induction of TSG-6 by IL-1 could be distinguished from 25 mmol/L D-glucose stimulation of TSG-6 mRNA by the insensitivity of the former to protein synthesis inhibition by cycloheximide. This finding is similar to findings in vascular smooth muscle cells in which an IL-1 pathway could be distinguished from a growth-factor (epidermal growth factor/fibroblast growth factor-1) pathway by its insensitivity to protein synthesis inhibitors³³. Furthermore, a "growth factor" cycloheximide-sensitive pathway described in articular chondrocytes was also demonstrated to be delayed as compared with IL-1 stimulation³⁴. These data together with our findings demonstrate tight control of TSG-6 expression in numerous cell types via two distinct pathways.

In summary, our study demonstrates that proximal tubular cells express both the mRNA and protein of precursors of the II family of protease inhibitors. Furthermore, we show the inducible expression of TSG-6, which leads to an increase in plasmin inhibitory activity. Also shown are two distinct pathways in the proximal tubular cell by which TSG-6 synthesis may be stimulated. In conclusion, these observations suggest that stimuli that modulate the synthesis of the profibrotic cytokine TGF-1 may potentially modulate its activation.

References

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Acknowledgments

This study was supported by a stipend of the German Research Foundation (Deutsche Forschungsgemeinschaft; Graduiertenkolleg "Charakterisierung regulatorischer Peptide und ihrer Zielproteine," Hannover Medical School) to U.J. A.O.P. is supported by a Wellcome Trust Advanced Training Fellowship.