Macrophage-Stimulating Protein Is Produced by Tubular Cells and Activates Mesangial Cells
Teresa Rampino*,
Chiara Collesi,
Marilena Gregorini*,
Milena Maggio*,
Grazia Soccio*,
Paola Guallini* and
Antonio Dal Canton*
*Unit of Nephrology, Dialysis and Transplant, I. R. C. C. S. Policlinico San Matteo and University, Pavia, Italy; and Institute for Cancer Research and Treatment, Torino, Italy.
Correspondence to Dr. Antonio Dal Canton, Universita Degli Studi di Pavia, Unit of Nephrology, Policlinico San Matteo, 27100 Pavia, Italy. Phone: 39-0382-422037; Fax: 39-0382-525001; E-mail: dalcanton{at}mbox.medit.it
ABSTRACT. Until now, hepatocytes have been the only known cellsource of macrophage-stimulating protein (MSP), and tissue macrophageshave been the cells on which the biologic effects of MSP havebeen proved. To extend the understanding of the biologic meaningof MSP, it was investigated whether MSP operates in the kidney.MSP protein was evaluated by Western blot in supernatant ofcultured human tubular cells (HK2) and human mesangial cells(HMC). MSP mRNA was investigated in HK2 by reverse transcription–polymerasechain reaction (RT-PCR). The expression of the MSP receptor,RON, was evaluated in HMC and HK2 by Western blot. RON mRNAwas investigated in HMC by RT-PCR. The expression of MSP andRON in normal human renal tissue was studied by immunohistochemistry.HMC were stimulated with recombinant MSP (rMSP) and HK2 supernatantto study cell growth, migration, and the capacity to invadean artificial collagen matrix and synthesize interleukin-6 (IL-6).HK2 produced MSP and expressed RON in a form that was phosphorylatedby rMSP. HMC expressed RON but did not produce MSP. MSP in HK2supernatant and rMSP induced in HMC phosphorylation of RON,growth, migration, invasion, and IL-6 synthesis. In normal humankidney, tubules expressed MSP and RON. These results indicatea novel field of operation for MSP and suggest a pathogenicrole of the MSP/RON system in renal disease. In fact, MSP releasedby tubular cells may recruit monocytes/macrophages in inflammatorytubulointerstitial disorders. In addition, MSP either circulatingor as paracrine product may sustain glomerular mesangioproliferativedisease.
Macrophage-stimulating protein (MSP), originally described asa serum factor with chemotactic effect on resident macrophages(1–3), is an 80- to 95-kD protein that belongs to thefamily of plasminogen-related growth factors also known as scatterfactors (4,5). MSP is produced as a biologically inactive singlechain precursor (pro-MSP) that is cleaved by membrane-boundmacrophage proteases homologous to coagulation enzymes or bymembers of the kallikrein family to form a disulfide-linkedactive heterodimer (6,7).
The receptor for MSP is the product of the RON protooncogene,a tyrosine kinase that belongs to the human growth factor receptorfamily (8,9), which also includes human MET (10) and chickenSEA (11,12). MSP activates RON, inducing its tyrosine phosphorylation,which results in cell migration (13), shape change (7), andproliferation (14).
Until now, hepatocytes have been the only proved cell sourceof MSP (4,15), and the expression of RON has been documentedin resident peritoneal macrophages (16), granulocytes (14),keratinocytes (17), osteoclasts (18), epithelial tissues (19,20),and cell lines derived from hepatic, gastric, and mammary carcinomas(15,21–23). A role of the MSP/RON system in the physiologyof the kidney or in renal disease has never been studied. Infact, although hybridization of MSP cDNA to total mRNA extractedfrom renal tissue has been shown (4), we do not know whetherand which renal resident cells produce MSP. Furthermore, RONtranscripts have been observed in tubular epithelia during developmentof the mouse metanephric kidney (20), but the expression ofthe receptor in mature renal tissue or cultured renal cellshas never been explored.
In this study, we have investigated whether renal cells produceMSP or express RON and are the target of MSP. In view of thecommon epithelial origin of hepatocytes and tubular cells andof the functional similarity between macrophages and mesangialcells, we have hypothesized that tubular cells produce MSP andthat mesangial cells express RON and are activated by MSP. Theresults confirm our hypothesis and suggest that the MSP/RONsystem may operate in the kidney and play a pathogenic rolein tubulointerstitial inflammatory disorders and mesangioproliferativeglomerulonephritides.
Cell Cultures
Primary cultures of human mesangial cells (HMC) were establishedfrom normal kidney cortex of patients undergoing unilateralnephrectomy for renal cell carcinoma, as described elsewhere(24). HMC were cultured in RPMI 1640 medium (Sigma-Aldrich Co.,St. Louis, MO) supplemented with 20% fetal calf serum (SigmaAldrich), 2% L-glutamine (2 mM), 1% sodium pyruvate, and 1%penicillin/streptomycin (Life Technologies, Grand Island, NY)and maintained at 37°C in a 5% CO2 humidified atmosphere.For our experiments, HMC were used at passages 4 through 8.
Human renal tubular cell line HK2, human hepatoma cell lineHepG2, and human mammary carcinoma cell line T47D were purchasedfrom the American Type Culture Collection (Rockville, MD). HK2,HepG2, and T47D were cultured in RPMI 1640 medium supplementedwith 10% fetal calf serum and 1% penicillin/streptomycin andmaintained at 37°C in a 5% CO2 humidified atmosphere.
In some experiments, HMC were incubated with HK2 supernatantor with HK2 supernatant preincubated for 2 h with neutralizinggoat polyclonal anti-human MSP antibody (Ab; 2 µg/ml)(R&D Systems, Minneapolis, MN) without fresh medium.
MSP Protein Production by HK2 and HMC
The release of MSP in HK2 and HMC cell culture supernatant wasevaluated by immunoprecipitation and Western blot, by use ofa goat polyclonal anti-MSP Ab (R&D Systems) that recognizesboth the monomeric inactive form of MSP (pro-MSP) and the -chainof the active dimeric form. A 50 ml volume of supernatant wasincubated with 2 µg/ml of anti-MSP antibody (Ab) adsorbedto 20 µl of protein A–sepharose 4B packed beads(Pharmacia, Uppsala, Sweden). The same amount of HepG2 supernatantwas used as a positive control. Immunoprecipitates were washedwith an ice-cold buffer that contained 20 mM Tris-HCl (pH 7.4),150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 10% glycerol,1% Triton X-100, and inhibitors of proteases (aprotinin 10 µg/ml,pepstatin 10 µg/ml, leupeptin 50 µg/ml, soybeantrypsin inhibitor 100 µg/ml, and phenylmethanesulfonylfluoride 1 mM) (Sigma-Aldrich) and boiled after addition of2x sample buffer. After 10% sodium dodecyl sulfate–polyacrylamidegel electrophoresis was done (BioRad Laboratories, Richmond,CA), proteins were transferred to a nitrocellulose membrane(Amersham, Amersham, UK), and MSP was detected with the anti-MSPAb. Mouse anti-goat IgG conjugated with horseradish peroxidase(Sigma-Aldrich) was used as the secondary Ab. Enhanced chemiluminescencewas used for visualization of the peroxidase complex (PierceChemical Company, Rockford, IL).
MSP mRNA Expression in HK2
In these experiments, HepG2 cells known for their constitutivecapacity to produce a high amount of MSP (15) were used as controls.MSP mRNA expression was evaluated by reverse transcription followedby PCR (RT-PCR). Total RNA extracted from HK2 and HepG2 withguanidinium thiocyanate as described in Chomczynsky and Sacchi(25) was used for RT. The 20 µl reaction mixture contained2 µl of 10x reaction buffer (Bethesda Research Laboratories,Bethesda, MD), 1 µg of RNA, 50 U of RNAsin (Promega Biotec,Madison, WI), 2 µg random primers (Bethesda Research Laboratories),1 mM (each) dNTP, 5 mM of MgCl2 (New England Biolabs), and 25U of M-MLV reverse transcriptase (Bethesda Research Laboratories).The reaction mixture was incubated at 42°C for 1 h, thenthe enzyme was denatured for 5 min at 99°C, and the productswere kept at -20°C. PCR was carried out on the productsof the RT reaction as follows: in a final volume of 50 µl,the reaction mixture contained 5 µl of the products ofthe RT reaction as the template, 200 µM (each) dNTP, 15pmol of the 5' PCR primer, 15 pmol of the 3' PCR primer, and5 µl of Taq polymerase buffer 10x supplemented with 15mM MgCl2 and 2.5 U of Taq polymerase (New England Biolabs).Then, 40 cycles of denaturation, annealing, and extension wereperformed by use of a programmable thermal cycler controller.Denaturation was at 94°C for 2 min for the first cycle and1 min for subsequent cycles. The annealing temperature was 62.5°Cfor 1 min. The extension temperature was 72°C for 1 min,and the final extension was for 7 min at 72°C. The oligomersused for PCR amplifications were designed on the MSP cDNA sequenceas follows: sense oligomer corresponding to nucleotides 886to 906 (5'-AATACCACCACTGCGGGCGT-3') and antisense oligomer correspondingto nucleotides 1555 to 1576 (5'-TCAGTATCCACTGCTCCTTCA-3'). Theglyceraldehyde 3-phosphate dehydrogenase gene (G3PDH) mRNA servedas a control. Primers for the G3PDH gene were as follows: senseoligomer (5'-TGGTATCGTGGAAGGACTCATGAC-3') and antisense oligomer(5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'); they amplified a 190-bp product.RT-PCR for MSP and G3PDH were made separately and resolved on2% and 3% agarose gels (Sigma Aldrich), respectively.
Expression and MSP-Induced Phosphorylation of RON in HMC and HK2
The expression of RON was studied in HMC and HK2 by immunoprecipitationfollowed by Western blot. Because the activation of RON by MSPis associated with autophosphorylation of tyrosine residue inthe receptor, we studied the activation of RON by a tyrosinephosphorylation assay. We used as the control T47D cells, i.e.,a mammary carcinoma cell line that constitutively expressesRON and is activated by MSP.
Subconfluent HMC, HK2, and T47D cells were treated with recombinantMSP (rMSP; 50 ng/ml) (R&D Systems) for 10 min at 37°C.Cells were washed twice with cold phosphate-buffered saline(PBS) and lysed with an ice-cold buffer that contained 10 mMPIPES (pH 6.8; Sigma-Aldrich), 100 mM NaCl, 5 mM MgCl2, 300mM sucrose, 5 mM ethyleneglycos-bis(ß-aminoethyl ether)-N-N'-tetraaceticacid (DIM buffer) (Sigma-Aldrich), 1% Triton X-100, 100 µMsodium orthovanadate, and inhibitors of proteases (see above).The cell lysates were cleared by centrifugation at 15,000 xg for 15 min at 4°C; an equal amount (800 µg) of totalprotein extracts from each cell line, determined by use of theBCA Protein Assay Reagent Kit (Pierce Chemical Company), wasimmunoprecipitated by stirring for 2 h at 4°C with the specificanti-RON C-terminal antisera adsorbed to 20 µl of proteinA–sepharose 4B packed beads. The immunocomplexes werewashed with lysis buffer, and proteins from immunoprecipitateswere solubilized in boiling Laemmli buffer in reducing conditions(26). The proteins were separated on 7% sodium dodecyl sulfate–polyacrylamidegel electrophoresis and transferred to nitrocellulose Hybondfilters. Filters were probed with mouse anti-phosphotyrosinemAb (U.B.I., Lake Placid, N.Y.) and anti-RON polyclonal Ab (producedin the Institute for Cancer Research and Treatment, Torino,Italy) (14); the specific binding was detected by the enhancedchemiluminescence system ECL-Plus.
RON mRNA Expression in HMC
The expression of RON mRNA in HMC was evaluated by RT-PCR. T47Dcells were used as the control. Total RNA was used for RT. The40 µl reaction mixture contained the enzyme buffer, 2µg of RNA, 1 U/µl of RNAsin, 50 pmol of the 3' PCRprimer (see below), 1 mM (each) dNTP, and 10 U/µl of M-MLVreverse transcriptase. The reaction was incubated at 37°Cfor 1 h; the enzyme was then denatured for 3 min at 95°Cand the products kept at -20°C. PCR was carried out on theproducts of the RT reaction as follows: in a final volume of100 µl, the reaction mixture contained 10 µl ofthe products of the RT reaction as source of the template, 50mM Tris (pH 8.4), 50 mM KCl, 25 µg/ml bovine serum albumin,2 µmol (each) dNTP, 35 pmol of the 5' PCR primer, 35 pmolof the 3' PCR primer, and a variable concentration of MgCl2.Then, 5 U of Taq polymerase were added and 100 µl of mineraloil were overlaid on the reaction mixture, and 30 cycles ofdenaturation, annealing, and extension were then performed byuse of a programmable thermal cycler controller. Denaturationwas at 92°C for 5 min for the first cycle and for 1 minfor subsequent cycles. The annealing temperature was 55°Cfor 1 min. The extension temperature was 72°C for 2 min.The extension time was calculated under the assumption of arate of extension of 1000 bases/min, according to the predictedlength of the amplified product.
The oligomers used for PCR amplifications were designed on theRON cDNA sequence as follows: sense oligomer corresponding tonucleotides 3236 to 3257 (5'-GTCAAGGATGTGCTGATTCCC-3') and antisenseoligomer corresponding to nucleotides 4366 to 4389 (5'-TCTGTGGAGTGAGGTACCTAATG-3').The G3PDH mRNA served as the control. RT-PCR for RON and G3PDHwere made separately and resolved on 1% and 3% agarose gels,respectively. The bands were transferred into a nylon membraneHybond N, and the specific binding was detected by use of aDirect Nucleic Acid Labeling System Kit (Amersham) as instructedby the supplier.
Effects of MSP on HMC Cell Growth Assay.
HMC (2 x 104 cells/well) were incubated for 24 and 48 h withrMSP at scalar concentrations (0, 10, 30, and 50 ng/ml), HK2supernatant, and HK2 supernatant preincubated with neutralizinganti-MSP polyclonal Ab. Viable cells were determined by trypanblue assay. Growth was evaluated by use of a Cell Titer 96 assay(Promega Biotec, Madison, WI). The assay is based on a colorimetricmethod (27) to determine the number of viable cells. Experimentswere repeated six times with each dose of rMSP and HK2 surnatant.
Motility and Matrix Invasion Assay.
The motility and matrix invasion assays (28) were performedin Transwell chambers (Costar Corporation, Cambridge, Mass).Cells were seeded on the upper side of a porous polycarbonatemembrane (8-µm pore size) coated (cell invasion assay)or not coated (cell migration assay) with an artificial basementmembrane that consisted of collagen type IV, laminin, and glycosaminoglycans(Matrigel [12.5 µg per filter], Becton Dickinson, Bedford,MA). Bottom wells were filled with 500 µl of completemedium supplemented either with rMSP (50 ng/ml), with HK2 supernatant,or with HK2 supernatant preincubated with anti-MSP Ab. After24 (for the migration assay) or 48 (for the invasion assay)h of incubation, the cells that remained on the upper side ofthe filters were mechanically removed, and the cells that migratedor invaded the Matrigel, passing into the lower side, were countedwith a Burker chamber. Experiments were repeated six times witheach dose of rMSP and HK2 supernatant.
Interleukin-6 Assay.
Soluble interleukin-6 (IL-6) in HMC supernatants was assayedby use of a commercial enzyme immunotest kit (R&D Systems),according to the manufacturer’s procedure. The sensitivityof the enzyme-linked immunosorbent assay system was 0.7 pg/ml.Experiments were repeated four times.
Effect of MSP on HK2 cell growth
HK2 cells (2 x 104 cells/well) were incubated for 24 and 48h with rMSP at scalar concentrations (0, 3, 5, 10, 30, and 50and ng/ml). Cell growth was evaluated by counting cells in aNeu Bauer chamber. Experiments were repeated six times witheach dose of rMSP.
In Vivo Expression of MSP and RON in Human Renal Tissue
The expression of MSP and RON in human kidney was evaluatedby immunohistochemistry. Normal human renal tissue was obtainedfrom kidney excised for renal cancer. The specimens were fixedin formalin acetate 6% for 24 to 48 h, then embedded in paraffinwax and serially sectioned (3 µm). The sections were dewaxedin xylol, passed in a decreasing series of alcohol, and finallyrehydrated with distilled water. Endogenous peroxidase was blockedwith H2O2 3.7% vol/vol in H2O for 15 min. After three washingsin 150 mM PBS (Sigma-Aldrich), the sections were exposed toanti-MSP monoclonal Ab (R&D Systems, diluted 1:300 in PBS/bovineserum albumin 1% overnight at 4°C) or to anti-RON monoclonalantibody (Transduction Laboratories, Lexington, KY, diluted1:750 in PBS/bovine serum albumin 1% overnight at 4°C).The secondary Ab and the complex streptavidin-biotin-peroxidasesteps were performed according to manufacturers of the LSAB+Kit (Dako, Glosrup, Danmark). Visualization was in 3,3 diaminobenzidine(Dako). Harris hematoxylin (Dako) was used to counterstain thenuclei lightly. Finally, the sections were dehydrated in increasingalcohol scale (95° to 100°, xylol), and the coverslipwas mounted with synthetic nonaqueous mounting media (Dako)for analysis with a ZEISS microscope (10x, 20x, and 40x).
Statistical Analyses
ANOVA and the Tukey Kramer test were used for comparison ofthe means.
HK2, but Not HMC, Produce MSP Figure 1 illustrates the results of the immunoprecipitationand Western blot performed with anti-MSP Ab in supernatant ofHK2, HMC, and HepG2 cells. A distinct couple of bands of 85and 55 kD, corresponding to pro-MSP and the MSP -chain, respectively,is apparent both in HK2 and in HepG2 supernatant but is absentin HMC supernatant.
Figure 1. Western blot performed with anti–macrophage-stimulating protein (MSP) antibody (Ab) in culture supernatant of the human tubular cell line HK2, human mesangial cells (HMC), and the hepatoma cell line HepG2. A 50 ml volume of supernatant was immunoprecipitated with anti-MSP polyclonal Ab adsorbed to protein A–sepharose 4B packed beads. Immunoprecipitates were washed with an ice-cold buffer, boiled after addition of 2x sample buffer, and the proteins were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were analyzed by Western blot that used a polyclonal anti-MSP Ab. The 85-kD bands represent monomeric MSP (pro-MSP), and the 55-kD bands represent the -chain of dimeric (active) MSP. Both HK2 and HepG2 cells (used as controls) release pro-MSP that is cleaved into the dimeric form in the supernatant. No band is visible in the HMC lane.
The expression of MSP mRNA in HK2 and HepG2 cells, evaluatedby RT-PCR, is shown in Figure 2. The large 669-bp band, correspondingto MSP cDNA in the HepG2 lane, confirms that HepG2 cells arestrong producers of MSP. A well-defined band of 669 bp is presentalso in the HK2 lane, which indicates that these cells alsoexpress MSP mRNA.
Figure 2. Expression of MSP mRNA in HK2 and HepG2 cells, determined by reverse transcription (RT)–PCR that used 1 µg of RNA as the template. The 669-bp band corresponds to MSP cDNA and indicates that MSP is transcribed in HK2 and HepG2 cells. The latter cell line is a known producer of MSP and was used as the control. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) is the RT-PCR control.
MSP Is Expressed in Tubules of Normal Kidney
The results of immunohistochemistry performed with anti-MSPantibody are illustrated in Figure 3. The tubules express MSPwith a fine granular cytoplasmatic distribution, whereas glomerulishow negative staining. MSP is expressed in all tubular segmentsin uniform and diffuse mode.
Figure 3. Immunohistochemical staining for MSP in normal human renal tissue. The diffuse brown staining of tubules demonstrates tubular localization of MSP. No staining is present in glomeruli. Magnification, x40.
RON Is Expressed in HMC as a Functionally Active MSP Receptor
RON mRNA expression was investigated by RT-PCR in HMC and inT47D cells (used as controls). Figure 4 shows, in the T47D lane,the expected 1153-bp band representing RON cDNA. The 1153-bpband present in the HMC lane indicates that these cells alsoexpress constitutively the RON transcript.
Figure 4. RON mRNA expression in HMC and the mammary carcinoma cell line T47D, determined by RT-PCR that used 1 µg of RNA as the template. The 1153-bp band corresponding to RON cDNA indicates transcription of RON by HMC and T47D cells. The latter cell line is known to express constitutively RON mRNA and was used as the control. G3PDH mRNA was the RT-PCR control.
The expression of RON protein in HMC and its tyrosine phosphorylationin response to stimulation with rMSP are shown in Figure 5:in the lower panel, immunoprecipitation of cell lysate withanti-RON Ab and subsequent Western blot with the same Ab identifya large 150-kD band (p150Ron) that corresponds to RON in T47Dcells both treated (+) and not treated (-) with rMSP; althoughless marked than in T47D, the band that represents RON is alsowell visible in HMC. In the upper panel, immunoprecipitationwith anti-RON Ab and subsequent Western blot with anti-phosphotyrosineAb identify in T47D and HMC treated with rMSP (+), but not incells not treated (-), a 150-kD band (p150Ron) overlapping withthe RON band in the lower panel. Thus, in HMC the RON kinaseis constitutively expressed and is functionally active, becauserMSP triggers its tyrosine phosphorylation.
Figure 5. Expression of RON on HMC as a functionally active receptor. (Lower panel) Immunoprecipitation (Ip) of 800 µg of total protein extracts from HMC and T47D lysate with anti-RON Ab, followed by Western blot (Wb) performed with the same anti-RON Ab (anti-RON), identified a large 150-kD band representing the RON protein (p150Ron) in T47D cells, which were used as controls. The p150Ron band is also visible in HMC. Expression of the RON receptor is independent of pretreatment with recombinant MSP (MSP) (+, treated cells; -, untreated cells). (Upper panel) Ip of 800 µg of total protein extracts from HMC and T47D lysate with anti-RON Ab, followed by Wb performed with anti-phosphotyrosine Ab (anti P-Tyr) identified a band, overlapping with the p150Ron band in the lower panel, representing phosphorylated RON. Phosphorylated RON is present only in T47D and HMC treated (+) with MSP. The 170-kD band (p170Ron) visible in the lower panel is the RON precursor that is recognized by the anti-RON Ab used both for immunoprecipitation and Western blot. The band is not visible in the upper panel because the anti P-Tyr Ab used for Western blot recognizes phosphotyrosine that is present only in the final activated form of RON.
RON is Expressed in HK2 as a Functionally Active MSP Receptor
The expression of the RON receptor in HK2 and its phosphorylationinduced by rMSP are shown in Figure 6: in the lower panel, immunoprecipitationand subsequent Western blot with the same anti-RON Ab identifythe 150-kD band (p150Ron) corresponding to RON in T47D (control)and HK2 cells both treated (+) and not treated (-) with rMSP.In the upper panel, immunoprecipitation with anti-RON Ab andsubsequent Western blot with anti-phosphotyrosine Ab identifyin T47D and HK2 treated with rMSP (+), but not in cells nottreated (-), the 150-kD band (p150Ron) overlapping with theRON band in the lower panel. Thus, in HK2 cells the RON kinaseis constitutively expressed and is functionally active.
Figure 6. Expression of RON on human tubular cell line HK2 as a functionally active receptor. (Lower panel) Ip of 800 µg of total protein extracts from HK2 and T47D lysate with anti-RON antibody, followed by Wb performed with the same anti-RON Ab (anti-RON), identified a large 150-kD band representing RON protein (p150Ron) in T47D cells, which were used as controls. The p150Ron band is also visible in HK2. Expression of the RON receptor is independent of pretreatment with MSP (+, treated cells; -, untreated cells). (Upper panel) Ip of 800 µg of total protein extracts from HMC and T47D lysate with anti-RON Ab, followed by Wb performed with anti P-Tyr identified a band, overlapping with the p150Ron band in the lower panel, representing phosphorylated RON. Phosphorylated RON is present only in T47D and HK2 treated (+) with MSP.
MSP Triggers Biologic Responses in HMC and HK2 Figure 7 shows that rMSP stimulates mesangial cells growth ina time- and dose-dependent manner and that HK2 supernatant stimulatesHMC growth in a time-dependent manner. The latter effect isblocked by the addition in the HK2 supernatant of anti-MSP Ab.
Figure 7. Human mesangial cells were conditioned with rMSP at scalar concentrations, with supernatant of tubular cell culture (HK2 sn) and with HK2 sn plus neutralizing anti-MSP Ab (anti-MSP pAb). Columns are means of six experiments (bars, SD). Both rMSP and MSP in supernatant induced a time-dependent and dose-dependent growth of mesangial cells. *P < 0.05 versus fetal calf serum (FCS); #P < 0.01 versus FCS; °P < 0.05 versus HK2sn + anti-MSP pAb; P < 0.01 versus HK2 sn plus anti-MSP pAb.
Figure 8A shows the chemotactic effect of rMSP and HK2 supernatanton HMC. In fact, all the cells migrate from the upper well wherethey are seeded into the lower well, which contains rMSP orHK2 supernatant, and cell migration is abrogated by anti-MSPAb.
Figure 8. HMC (2 x 104) were seeded in the upper side of Transwell chambers not coated (migration assay, A) or coated (invasion assay, B) with Matrigel. Bottom wells were filled with FCS, rMSP (50 ng/ml), rMSP (50 ng/ml) plus anti-MSP pAb (2 µg/ml), HK2 sn, and HK2 sn plus anti-MSP pAb. Columns are means of six experiments (bars, SD). Black columns represent cells that do not migrate to the lower well and remain in the upper well, and white columns represent cells that migrate into the lower well. rMSP and HK2 supernatant induce in vitro a chemotactic effect and an invasive phenotype in HMC. Neutralizing polyclonal anti-MSP Ab abrogates cell migration and invasion.
Figure 8B shows that HMC stimulated with rMSP and HK2 supernatantinvade an artificial basement membrane. In fact, all the cellspass through the Matrigel layer and migrate into the lower wellthat contains rMSP or HK2 supernatant. Again, anti-MSP Ab abrogatesthe phenomenon.
Figure 9 shows that rMSP stimulates HMC to release IL-6 in atime-dependent manner. Figure 10 shows that rMSP stimulateshuman tubular cell growth in a time-dependent and dose-dependentmanner.
Figure 9. Effects of MSP on interleukin-6 (IL-6) release by HMC. HMC were stimulated with rMSP (50 ng/ml) for 24 and 48 h. Columns are means of four experiments (bars, SD). rMSP induced a time-dependent IL-6 release by HMC. *P < 0.001 versus FCS; °P < 0.001 versus rMSP at 24 h.
Figure 10. Effect of rMSP on HK2 cells. Columns are means of six experiments (bars, SD). rMSP induced a dose-dependent and time-dependent growth of HK2. *P < 0.005 and °P < 0.001 versus FCS, 24 h; P < 0.01 and #P < 0.001 versus FCS, 48 h.
Ron Is Expressed in Glomeruli and Tubules of Normal Kidney Figure 11 illustrates the results of immunohistochemistry studyperformed with anti-RON antibody. In glomeruli, RON is expressedin distinct mesangial cells. In addition, RON is diffusely anduniformly expressed in the tubules.
Figure 11. Immunohistochemical staining for RON in normal human renal tissue. Distinct mesangial cells stain positive for RON (in brown) in the glomerular tuft. In addition, RON is uniformly expressed in the tubules. Magnification, x40.
MSP was originally identified as a serum protein that stimulatesmurine peritoneal resident macrophages to make a chemotacticresponse to C5a (1,3). MSP also alters the morphology of adherentmacrophages, stimulates ingestion of C3bi-coated erythrocytes(3), and inhibits expression of inducible nitric oxide synthasemRNA in endotoxin-stimulated or cytokine-stimulated macrophages(29). Northern blotting of human tissue RNAs showed that liveris the major locus of MSP mRNA (4). MSP circulates in the bloodas a single-chain precursor that is cleaved into an active heterodimerby enzymes of the coagulation cascade and by macrophage membraneproteases (6,7). The receptor for MSP is the product of theRON protooncogene, which is expressed in macrophages (16), keratinocytes(17), adherent monocytes (14), and a variety of tumoral cells(15,21–23). The biologic significance of the MSP/RON systemremains largely unknown. In view of the effects of MSP on macrophagesand keratinocytes, it is supposed that MSP produced in the liverand circulating in inactive form is activated in wounds by coagulationfactors and membrane-bound macrophage enzymes and plays a rolein inflammation and healing (30,31). In addition, its effectson cell replication and migration suggest an oncogenic potential(23,32). Information on sites of MSP production other than theliver is scanty. Northern blot analysis of a human multipletissue showed MSP mRNA expressed in the kidney (4). However,we ignore which renal cells transcribe the MSP gene and whetherthe MSP protein is actually produced in the kidney in vivo.Another unexplored point is whether there are renal cells thatbear the RON receptor and are the target of MSP.
In this study, we have investigated the renal expression ofthe MSP/RON system. In particular, in conceiving the study,we have addressed tubular cells as MSP producers, because theyare epithelial cells as the hepatocytes. In addition, we havehypothesized that mesangial cells, i.e., the specialized cellswith macrophagic functions resident in glomeruli, express theRON receptor, and respond to MSP.
Our results demonstrate that renal tubular cells produce MSP.In fact, cultured HK2 express MSP mRNA and release MSP proteinin their supernatant. The production of MSP by tubular cellsis confirmed in vivo by immunohistochemistry that shows positivestaining for MSP in tubules of normal renal tissue. The expressionof MSP in tubules is uniform and extended to all tubular segmentsand is specific for tubular cells, because no MSP staining ispresent in glomeruli or in renal vessels in vivo, and mesangialcells do not release MSP in vitro. Of interest, MSP is presentin HK2 supernatant both as precursor and as the active dimericform. In fact, the supernatant contained the -chain of dimericMSP and induced in target (mesangial) cells biologic responsesthat were neutralized by anti-MSP Ab. Therefore, we supposethat MSP is secreted by HK2 cells as biologically inactive pro-MSPand is cleaved by proteases present in serum contained in culturemedium (6,7). Quite unexpectedly, we found that tubular cells,in addition to being producers of MSP, bear the MSP receptorRON. In fact, RON is uniformly expressed in all tubular segmentsin vivo and is present in HK2 cells in vitro in a form thatis able to work, i.e., that can be phosphorylated by MSP. Anyspeculation about the physiologic meaning of the coexpressionof MSP and its receptor in renal tubules is hampered by thepaucity of information on the physiologic role of the MSP/RONsystem in general. Recent investigations have shown that MSPis a growth factor for keratinocytes that regulates their integrin-dependentadhesion to extracellular matrix and prevents apoptosis of keratinocytesand MDCK cells transfected with RON cDNA (33,34). In this study,we found that MSP stimulates tubular cell growth. These findingssuggest that MSP participates as an autocrine factor in theregulation of renal tubular cell growth and survival.
Another original finding of our study is that glomerular mesangialcells express RON both in vivo and in vitro and that the mesangialreceptor is functionally active. In fact, rMSP induces the phosphorylationof RON on mesangial cells and causes important biologic effects,i.e., growth, migration, invasion into an artificial collagenmatrix, and synthesis of IL-6. The observations that MSP isproduced by renal tubules and is active on mesangial cells suggesta role of MSP/RON in renal pathophysiology and offer a new perspectiveto look at the biologic meaning of the system. In fact, MSPreleased by tubular cells in the course of tubulointerstitialinflammatory disorders, like interstitial nephritis or renalgraft rejection, once activated locally, e.g., by coagulationfactors, may stimulate resident macrophages and recruit circulatingmonocytes, thus amplifying the inflammatory response. Supportingthis hypothesis is our preliminary observation that circulatingmonocytes treated with lipopolysaccharide or inflammatory cytokinesexpress de novo RON (35). In addition, the finding that mesangialcells express RON and, when treated with MSP, proliferate, migrate,become invasive, and synthesize IL-6 suggests a role of MSP/RONin mesangioproliferative and membranoproliferative glomerulonephritides.In fact, in this form of glomerular disease, mesangial cellsproliferate and move into the subendothelial space, causingprogressive expansion of the mesangium and obliteration of glomerularcapillaries. In addition, IL-6 is a growth factor for mesangialcells (36). MSP may reach the mesangial cells in vivo, eitherfrom the circulation or as a paracrine factor released by tubularcells, and find locally activating proteases. In fact, glomerulardeposition of coagulation products is a current finding in thecourse of glomerular inflammation (37).
In summary, our study gives evidence that renal tubular cellsproduce MSP and express RON and that glomerular mesangial cellsexpress RON and are activated by MSP. These novel observationsextend the possible field of operation of MSP/RON to the kidneyand are the basis of further studies aimed at understandinga possible role of MSP/RON in renal physiology and inflammatorydisorders.
Acknowledgments
We thank Dr. P. Malvezzi for his technical assistance.
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Received for publication April 17, 2001.
Accepted for publication September 14, 2001.
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Abstract
Introduction
-chain of the active dimeric form. A 50 ml volume of supernatant was incubated with 2 µg/ml of anti-MSP antibody (Ab) adsorbed to 20 µl of protein A–sepharose 4B packed beads (Pharmacia, Uppsala, Sweden). The same amount of HepG2 supernatant was used as a positive control. Immunoprecipitates were washed with an ice-cold buffer that contained 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 10% glycerol, 1% Triton X-100, and inhibitors of proteases (aprotinin 10 µg/ml, pepstatin 10 µg/ml, leupeptin 50 µg/ml, soybean trypsin inhibitor 100 µg/ml, and phenylmethanesulfonyl fluoride 1 mM) (Sigma-Aldrich) and boiled after addition of 2x sample buffer. After 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis was done (BioRad Laboratories, Richmond, CA), proteins were transferred to a nitrocellulose membrane (Amersham, Amersham, UK), and MSP was detected with the anti-MSP Ab. Mouse anti-goat IgG conjugated with horseradish peroxidase (Sigma-Aldrich) was used as the secondary Ab. Enhanced chemiluminescence was used for visualization of the peroxidase complex (Pierce Chemical Company, Rockford, IL). 
P < 0.01 versus HK2 sn plus anti-MSP pAb.
