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J Am Soc Nephrol 11:1620-1630, 2000
© 2000 American Society of Nephrology

Activation of DNA Synthesis and AP-1 by Profilin, an Actin-Binding Protein, via Binding to a Cell Surface Receptor in Cultured Rat Mesangial Cells

MASAHITO TAMURA*,{dagger},{ddagger}, NOBUYUKI YANAGIHARA§, HIROSHI TANAKA*, AKIHIKO OSAJIMA*, TAKESHI HIRANO||, KEN HIGASHI{dagger}, KENNETH M. YAMADA{ddagger}, YASUHIDE NAKASHIMA* and HIDEYASU HIRANO{dagger}

* Second Department of Internal Medicine, Institute of Industrial Ecology, University of Occupational and Environmental Health, Kitakyushu, Japan
{dagger} Department of Biochemistry, Institute of Industrial Ecology, University of Occupational and Environmental Health, Kitakyushu, Japan
§ Department of Pharmacology, School of Medicine, Institute of Industrial Ecology, University of Occupational and Environmental Health, Kitakyushu, Japan
|| Department of Environmental Oncology, Institute of Industrial Ecology, University of Occupational and Environmental Health, Kitakyushu, Japan
{ddagger} Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.

Correspondence to Dr. Masahito Tamura, Second Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu, 807-8555 Japan. Phone: 81-93-603-1611; Fax: 81-93-691-6913; E-mail: mtamura{at}med.uoeh-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Profilin is known to bind to actin monomers (to regulate actin polymerization) and to phosphatidylinositol-4,5-bisphosphate (to inhibit hydrolysis by unphosphorylated phospholipase C-{gamma}l). It was recently reported that profilin is overexpressed in glomerular mesangial cells (MC) of rats with anti-Thy-1.1-induced glomerulonephritis and is accumulated in the extracellular space around MC. In this study, the biologic activities of extracellular profilin were examined. Scatchard analysis indicated the existence of a single class of cell surface binding sites, with similar equilibrium dissociation constants for purified splenic profilin and recombinant profilin, in cultured rat MC. Profilin increased [3H]thymidine incorporation in a dose-dependent manner and produced additive effects on platelet-derived growth factor-induced [3H]thymidine incorporation. Profilin increased AP-1 DNA-binding activity in a concentration-dependent (ED50 = 30 nM) and time-dependent manner after transient c-jun gene expression, as measured using gel-shift assays and competitive reverse transcription-PCR. Pretreatment of profilin with an anti-profilin inhibitory antibody suppressed profilin-induced AP-1 activation and [3H]thymidine incorporation. Furthermore, profilin induced rapid transient activation of protein kinase C, and staurosporine and H-7 reduced the profilin-induced activation of AP-1, suggesting protein kinase C-dependent activation of AP-1. These findings indicate that profilin in the extracellular space can bind to cell surface receptors of MC and act as an inducer of signal transduction. These results suggest that extracellular profilin may be involved in the progression of glomerular diseases, by affecting cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Profilin is a conserved and widely distributed cytoplasmic protein in eukaryotic cells that binds actin monomers in a stable 1:1 complex (1,2) and affects the growth or dissolution of actin filaments by serving as a reversible storage reservoir for actin monomers (3). In addition to actin, several ligands for profilin have been well characterized in vitro, including phosphatidylinositol-4,5-bisphosphate (PIP2), poly(L-proline), and vasodilator-stimulated phosphoprotein (VASP) (3,4,5). Its interactions with signaling pathway components such as PIP2 place profilin at the crossroads of transmembrane signal transduction and regulation of the actin cytoskeleton (4). Profilin is expressed in all eukaryotic cells examined to date (6,7,8), including mesangial cells (MC), and its necessity for life indicates that profilin is important in cell function (9). Although the expression of profilin is usually constitutive, profilin levels can be increased in some pathologic states, including liver, gastric, and kidney diseases (8,10,11).

Numerous studies have shed light on the intracellular role of profilin; however, in a variety of clinical situations, profilin, actin, and other actin-binding proteins are also released into the extracellular space during severe cell damage, including cell lysis (12). Anti-Thy 1.1-induced glomerulonephritis is a rat model of human mesangial proliferative glomerulonephritis associated with severe MC injury and mesangiolysis, followed by MC proliferation (13), suggesting that profilin might be released into the extracellular space after MC damage. In fact, we demonstrated that profilin is overexpressed in MC of rats with anti-Thy-1.1-induced glomerulonephritis and accumulates in the extracellular space around the plasma membrane of MC (8,14). In this model and in a variety of human progressive glomerular diseases, mesangial injury and mesangiolysis are observed (15,16,17). The elevated levels of profilin released from dying cells could conceivably induce intracellular signaling in surrounding cells if some type of receptor for profilin exists.

The biologic activity of profilin present in the extracellular space has not been characterized. Therefore, in this study, we examined whether cell surface receptors for profilin might exist and, if so, whether extracellular profilin could affect transcription factors, such as AP-1, and DNA synthesis via such receptors. AP-1 is a key element in signal transduction in response to stimuli such as cytokines, leading to cell growth. AP-1 consists of homoor heterodimeric protein complexes formed by the related jun and fos gene family members. It binds to a consensus sequence known as the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE), which controls transcription of a variety of genes and cell growth (18), suggesting that AP-1 plays important roles in the pathogenesis of renal diseases (13). This study establishes a novel function for profilin; it specifically binds to a putative receptor on MC and stimulates DNA synthesis and AP-1 DNA-binding activity in MC. These results suggest that profilin may affect the progression of glomerular diseases, possibly by regulating cell growth, when it is present in the extracellular space.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Poly(L-proline), cycloheximide, staurosporine, H-7, and platelet-derived growth factor-BB (PDGF-BB) were obtained from Sigma Chemical Co. (St. Louis, MO). Poly(dI-dC)·poly(dI-dC), random hexamers, murine Moloney leukemia virus reverse transcriptase, and dNTP were obtained from Pharmacia Biotech (Uppsala, Sweden). [{gamma}-32P]ATP was obtained from Amersham Life Science (Arlington Heights, IL), and affinity-purified polyclonal anti-c-Jun antibody (SC-45) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Cultures
Glomerular MC strains from male Sprague-Dawley rats were isolated by a differential sieving method and characterized as previously described (8,19). Rat MC were maintained at 37°C, in a 5% CO2 incubator, in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. MC were used between passages 5 and 10. To avoid any effects from profilin in the culture medium, cells were maintained for 24 h before experiments in medium that had been passed three times through a poly(L-proline)-Sepharose column, to remove all profilin.

Construction of an Expression Vector Encoding Profilin
The reverse transcription (RT)-PCR product (8,20) spanning the protein-coding region of rat profilin (from the initiator methionine to the termination codon) was subcloned into the BamHI/HindIII site of the expression vector pQE30 (Qiagen, Valencia, CA) and was confirmed by sequencing; the plasmid was designated pRPROF. Expression of His6-profilin was induced by incubating transformed Escherichia coli with 1 mM isopropyl ß-D-galactopyranoside for 4 h, as described (21).

Purification of Profilin
Profilin was purified from the cytosolic fraction of bovine spleen and from E. coli cell sonicates carrying recombinant pRPROF by poly(L-proline) affinity chromatography (22), modified as follows. Spleen extracts were prepared by homogenizing 1 vol of spleen in 5 vol of ice-cold 10 mM Hepes buffer (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 10 µg/ml leupeptin, and 10 µg/ml pepstatin (23). The extract was centrifuged at 2000 x g for 10 min at 4°C. Triton X-100 (1%) and glycerol (2.5%) were added to the pooled supernatants, and the mixture was then centrifuged at 100,000 x g for 30 min at 4°C. The supernatant was filtered through a sintered glass filter and loaded onto a poly(L-proline)-Sepharose 4B column. After extensive washing with buffer A (10 mM Tris-HCl, pH 7.8, 0.1 M KCl, 0.1 M glycine), bound profilin was eluted with buffer A containing 30% DMSO. The fractions containing profilin were concentrated and dialyzed against phosphate-buffered saline (PBS) at 4°C before use.

The purity of profilin was confirmed as follows. Purified profilin was mixed with loading buffer containing 25 mM Tris-HCl (pH 6.8), 0.5% sodium dodecyl sulfate (SDS), 15% glycerol, 8 mM DTT, 2.5% 2-mercaptoethanol, and 8.3 M urea and was boiled for 10 min, and then 10 mM iodoacetamide was added. The samples were electrophoresed on SDS-containing 8 to 18% polyacrylamide gradient gels (Amersham Pharmacia Biotech, Piscataway, NJ). The gels were stained using the silver staining method or were blotted onto a polyvinylidene fluoride membrane, incubated with 500 ng/ml antiprofilin antibody [affinity-purified rabbit polyclonal antibody raised against synthesized profilin oligopeptide (phenylalanine-84 to serine-92, aligned with bovine profilin), which recognizes rat, mouse, and bovine profilins but not human profilin (8)], reacted with peroxidase-conjugated goat anti-rabbit IgG F(ab')2 (diluted 1/1000; Cappel, Organon Teknika, Turnhout, Belgium), and then observed using an enhanced chemiluminescence method (ECL; Amersham Life Science). Recombinant profilin was also purified, using the same procedure, from sonicates of E. coli that had been transformed with pRPROF and induced with isopropyl ß-D-galactopyranoside.

Binding Studies with 125I-Profilin
Splenic and recombinant profilins were labeled with Na 125I (100 mCi/ml; DuPont/New England Nuclear, Boston, MA) and lactoperoxidase, using a labeling kit (DuPont) (24). 125I-Profilin was separated from unincorporated label on a Sephadex G-25 column and was purified with poly(L-proline). To measure total 125I-profilin binding to isolated rat MC, various concentrations of 125I-profilin (1 to 20 nM; specific activity, 91 cpm/fmol protein) were added to one set of microtubes, in 200 µl of Krebs-Ringer solution consisting of 137 mM NaCl, 5 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 12 mM NaHCO3, 15 mM Hepes, and 5 mM glucose (pH 7.4) with 0.1% bovine serum albumin. For measurement of nonspecific binding, an identical set of tubes was prepared and a 100-fold molar excess of unlabeled profilin was added. Binding reactions were initiated by addition of a MC suspension (3 to 5 x 106 cells/tube), and the tubes were incubated for 30 min at 4°C. The 125I-profilin bound to the cells was separated by centrifugation through silicon oil and dinonyl phthalate (1:1) at 12,000 rpm for 5 min, in a microfuge, and was counted in a gamma counter. The amount of 125I-profilin specifically bound to cells was calculated as the difference between total and nonspecific binding. The binding data were evaluated by Scatchard analysis (25).

Gel-Shift and Supershift Analyses
To detect binding to DNA elements, gel-shift analyses were performed using nuclear extracts prepared from profilin-treated or control cells as described (23), with the following modifications. Cells were seeded in 10-cm-diameter tissue culture dishes, grown to subconfluence, serum-starved for 24 h before incubation with profilin in medium containing 0.5% FCS, and incubated with or without 6.7 µM profilin for the indicated times. In the dose-dependence experiments, 1 nM to 10 µM profilin was present for 3 h. For the antibody inhibition study, 100 nM profilin was preincubated for 1 h at 37°C in culture medium containing the indicated concentrations of anti-profilin peptide antibody (8) and was then used in 3-h profilin stimulation assays. Cells were homogenized in ice-cold 10 mM Hepes buffer (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 µg/ml leupeptin, and 10 µg/ml pepstatin (23). The extract was centrifuged at 2000 x g for 10 min at 4°C. The pellets were resuspended in 20 mM Hepes buffer (pH 7.9) containing 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetate (EDTA), 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT and were then centrifuged for 30 min at 25,000 x g. The resulting supernatants were dialyzed for 5 h against 20 mM Hepes buffer (pH 7.9) containing 20% (vol/vol) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT and were used as nuclear extracts. Nuclear extracts equivalent to 5 µg of protein were incubated for 20 min at room temperature in a DNA-binding mixture containing 2.5 ng of poly(dI-dC)·-poly(dI-dC), 0.5 µl of 32P-labeled TRE oligonucleotide (106 cpm/pmol), 20 mM Tris-HCl (pH 7.5), 5% glycerol, 2.5 µg of bovine serum albumin, and 5 mM EDTA, in a final volume of 10 µl. The TRE sequence used for gel-shift analyses was 5'-ACAGGTGACTCACCTGGG-3' (the consensus DNA-binding sequence is underlined). The reactions were electrophoresed on native 6% polyacrylamide gels. The gels were then exposed to screens that chemically accumulate ß-emissions, which were observed using an image analyzer system (BAS 2000; Fuji, Tokyo, Japan). For supershift analyses, gel-shift analyses were performed with the addition of 1 µl of affinity-purified polyclonal anti-c-Jun antibody 2 h before the incubation.

RNA Extraction and RT-PCR
Poly(A)+ RNA was purified from control or profilin-treated MC using oligo(dT)-cellulose chromatography, as described (11,26). The first-strand cDNA was synthesized from 250 ng of poly(A)+ RNA in 50 mM Tris-HCl buffer (pH 8.3) containing 200 ng of random hexamers, 3 mM MgCl2, 400 U of murine Moloney leukemia virus reverse transcriptase, 500 µM dNTP, 15 mM DTT, and 75 mM KCl, in a final volume of 15 µl (1 h at 37°C). The cDNA obtained was amplified by PCR. Each sample was assayed for c-jun, c-fos, and ß-actin cDNA in separate tubes, using specific primers. The upstream and downstream primers for c-jun were 5'-ATGACTGCAAAGATGGAAACG-3' and 5'-TGCCGCGGAGGTGACACTGGG-3', respectively. PCR with these primers yielded a single band that corresponded to a 402-bp fragment that was identical to positions 353 to 754 in rat c-jun cDNA (27). The upstream and downstream primers for c-fos were 5'-ATGATGTTCTCGGGTTTCAAC-3' and 5'-AGGAGATAGCTGCTCTACTTT-3', respectively. These yielded a single band that corresponded to a 402-bp fragment that was identical to positions 189 to 590 in rat c-fos cDNA (28). The upstream and downstream primers for ß-actin were 5'-TGGAGAAGAGCTATGAGCTGCCTG-3' and 5'-GTGCCACCAGACAGCACTGTGTTG-3', respectively; they yielded a 201-bp PCR product, as described (29).

PCR was performed by incubating 250 ng of sample cDNA with 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 2.5 U of Taq DNA polymerase, 400 µM dNTP, and 40 pmol of primers, in a final volume of 50 µl. PCR using c-jun and c-fos primers was performed for 32 cycles (60 s at 94°C, 75 s at 58°C, and 90 s at 72°C), in an automatic thermal cycler (PC-800; Astec, Fukuoka, Japan). For ß-actin, we used 20 cycles (15 s at 94°C, 15 s at 65°C, and 30 s at 72°C). The final PCR mixture was electrophoresed using 6% polyacrylamide gel electrophoresis (PAGE); the gel was stained with ethidium bromide and quantified from the film negative by scanning densitometry (IBAS; Zeiss/Kontron Bildanalyse, Munich, Germany). To confirm the accuracy of the mRNA quantity amplified by RT-PCR, PCR was performed using differential PCR cycles (30 to 34 cycles for c-jun and c-fos and 18 to 22 cycles for ß-actin) or using incubations with serially increased amounts of glomerular mRNA. These controls revealed a dose- and cycle-dependent increase in the PCR product (data not shown).

Competitive RT-PCR
To determine the absolute amounts of target cDNA, we used an internal control that contained the same primer template sequences as the target (30,31). The competitor DNA was obtained by amplification of a foreign DNA fragment using two composite primers (20,30). Each composite primer had the target c-jun gene and prokaryotic chloramphenicol acetyltransferase gene primer sequences; the upstream forward primer (5'-ATGACTGCAAAGATGGAAACGTTTTCATCGCTCTGGAG-3') corresponded to positions 353 to 373 of the c-jun mRNA (underlined) and positions 4672 to 4650 of the pSV2CAT gene (italics). The reverse primer (5'-TGCCGCGGAGGTGACACTGGGGGCGAAGAAGTTGTCCAT-3') corresponded to positions 754 to 734 of the rat c-jun mRNA (underlined) and positions 4463 to 4483 of the pSV2CAT gene (italics). PCR was performed as described above, except that 1 µg of pSV2CAT DNA and these primers were added instead of sample cDNA and c-jun primers; in this way the designed length (243 bp) of the internal standard was confirmed. The amplified fragments were recovered electrophoretically and purified using phenol/chloroform and ethanol precipitation. The DNA fragment obtained included the c-jun primer template sequences on both sides of the fragment, but with a completely different intervening sequence and length.

All poly(A)+ RNA purified from profilin-treated (6.7 µM, 1 h) or control MC were reverse-transcribed and amplified with a competitor DNA. PCR was performed by incubating MC cDNA reverse-transcribed from 250 ng of poly(A)+ RNA and serially decreased amounts of competitor DNA with c-jun primers in the buffer described above. The reactions were performed for 32 cycles (60 s at 94°C, 75 s at 56°C, and 90 s at 72°C). After PCR, the amounts of products generated by the target and the internal control were compared using 5% PAGE. A standard curve was constructed by linear regression analysis, as described previously (31). The amount of competitor DNA yielding equimolar product identified the initial amount of the target gene.

Assay of Protein Kinase C (PKC) Activity
MC cultured to subconfluence in 5-cm-diameter tissue culture dishes were treated with or without 6.7 µM profilin for varying periods. Cytosolic and particulate fractions were prepared from the cells as described (32,33). PKC activity was measured as the incorporation of 32P into histone (type III-S) from [32P]ATP and was expressed as nanomoles of 32P incorporated per minute per milligram of protein.

Measurement of [3H]Thymidine Incorporation
To measure [3H]thymidine incorporation, MC were plated in 24-well dishes at a density of 1 x 104 cells/well, as described (34). After 48 h of serum deprivation (0.5% FCS), the cells were exposed to fresh medium containing 0.5% FCS, with or without the indicated concentrations of profilin. After 24 h of incubation, 1 µCi/ml [3H]thymidine was added to each well for 1 h. Cells were then washed three times with ice-cold PBS and collected by TCA precipitation. Incorporated radioactivity was determined using a liquid scintillation counter.

Statistical Analyses
Data are expressed as mean ± SD, except for the [3H]thymidine incorporation experiments (presented in Figure 3), in which the vertical bars represent SEM. Statistical evaluation of the data was performed using ANOVA or t test. Statistical significance was taken as P < 0.05.



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  		Figure 3. Effects of profilin on [3H]thymidine incorporation in MC. (A) Dose dependence of profilin effects on [3H]thymidine incorporation. Quiescent cultured rat MC were incubated for 24 h with increasing concentrations of splenic profilin. Data are presented as mean ± SEM of four independent experiments. *P < 0.01 versus control; **P < 0.005 versus control. (B) Effects of profilin on platelet-derived growth factor (PDGF)-induced activation of [3H]thymidine incorporation. Quiescent cultured rat MC were incubated for 24 h with or without 1 µM recombinant profilin or 10 ng/ml PDGF. Data are presented as mean ± SEM of three independent experiments. *P < 0.01 versus control; {dagger}P < 0.001 versus control; {ddagger}P < 0.01 versus PDGF alone. (C) Effects of anti-profilin antibody (Ab) on profilin-induced [3H]thymidine incorporation. Before the 24-h profilin treatment, splenic profilin (100 nM) was preincubated for 1 h at 37°C in culture medium with or without 10 µg/ml anti-profilin antibody. [3H]Thymidine incorporation was assayed as described in Materials and Methods. Data are presented as mean ± SEM of three independent experiments. *P < 0.01 versus control.

 


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of Profilin
Human (35), mouse (36), rat (8), and bovine (37) profilin cDNA have been reported. These mammalian profilins exhibit closely similar sequences, indicating that profilin is a highly conserved protein among mammals. Profilin is constitutively expressed in all eukaryotic cells examined to date, including splenic cells (6,7). We purified profilin from bovine spleen and from E. coli carrying the pRPROF expression vector for profilin, using poly(L-proline) affinity columns. After purification, SDS-PAGE with silver staining revealed a single 15-kD band (Figure 1). In immunoblotting, this 15-kD protein was recognized by the anti-profilin antibody. These observations confirmed that the protein obtained was profilin.



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  		Figure 1. Purified splenic and recombinant profilin. (A) Analytical gel for purified bovine splenic profilin used in this study. Bovine profilin, prepared as described in Materials and Methods, was resolved on a sodium dodecyl sulfate-containing 8 to 18% polyacrylamide gradient gel and was stained using the silver staining method (left lane). Molecular mass standards are indicated to the left of the gel. A duplicate gel was blotted onto a polyvinylidene fluoride membrane and incubated with 500 ng/ml affinity-purified rabbit anti-profilin antibody (right lane). (B) Silver staining of E. coli lysate carrying pRPROF (left lane) and recombinant profilin purified using a poly(L-proline) affinity column (right lane).

 

125I-Profilin Binding to MC
To investigate the plasma membrane receptor sites for extracellular profilin in rat glomerular MC, the cells were incubated with either splenic or recombinant 125I-profilin. As shown in Figure 2, 125I-profilin specifically bound to intact MC. Assays with higher concentrations of 125I-profilin (40 nM) demonstrated that binding was fully saturable and that 125I-profilin bound to cells was displaced by unlabeled profilin in a dose-dependent manner (data not shown). Scatchard analysis of the data yielded a linear plot, showing a single class of binding sites with a maximal binding capacity of 125 ± 9 fmol/5 x 106 cells and a equilibrium dissociation constant (Kd) of 34.3 ± 6.8 nM (Figure 2A). In terms of affinity, the recombinant profilin was similar to splenic profilin, with a Kd of 58.5 ± 17.5 nM (Figure 2B). The maximal binding capacity of the recombinant profilin (305 ± 102 fmol/5 x 106 cells) was only slightly higher than that of splenic profilin.



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  		Figure 2. Splenic and recombinant profilin binding to a cell surface receptor. (A) Splenic 125I-profilin binding to intact mesangial cells (MC) and Scatchard plot (inset). Cultured rat MC (3 to 5 x 106 cells/tube) were incubated with the indicated concentrations of 125I-profilin, as described in Materials and Methods. Binding parameters obtained from the negative inverse of the slope and the X-intercept are equilibrium dissociation constant (Kd) = 34.3 ± 6.8 nM and maximal binding capacity = 125 ± 9 fmol/5 x 106 cells, respectively (mean ± SD, duplicate determinations in four separate experiments). (B) Recombinant profilin binding and Scatchard plot (inset). Values of Kd = 58.5 ± 17.5 nM and maximal binding capacity = 305 ± 102 fmol/ 5 x 106 cells (mean ± SD) were determined from duplicate determinations in three separate experiments.

 

Effects of Profilin on [3H]Thymidine Incorporation
We studied the growth induction effect of extracellular profilin in vivo by examining [3H]thymidine incorporation using rat MC. [3H]Thymidine incorporation was increased in a dose-dependent manner (threshold, 10 nM; maximal induction, 1 µM) (Figure 3A). After 24-h exposure of quiescent MC to 1 µM profilin, [3H]thymidine incorporation increased from 10,089 ± 805 to 17,707 ± 952 cpm (1.8 ± 0.1-fold increase, P < 0.005 versus control). Next, we assayed for potential cooperative effects of profilin on MC proliferation with the growth factor PDGF, which is known to be released in many conditions involving MC injury (13). As shown in Figure 3B, recombinant profilin alone increased [3H]thymidine incorporation 1.9-fold, which was similar in magnitude to the increase produced by splenic profilin. Costimulation of cells with profilin and PDGF had an additive effect (from 3.9- to 4.9-fold), suggesting that profilin may increase cell proliferation by a mechanism different from that of PDGF and indicating that profilin has additive effects on PDGF-induced cell growth.

To exclude the possibility that increased [3H]thymidine incorporation might have resulted from contamination by growth factors during profilin preparation and to confirm that profilin specifically induces [3H]thymidine incorporation, we examined the effects of pretreatment of profilin with anti-profilin antibody. Before profilin stimulation of MC, profilin was preincubated with anti-profilin antibody. This pretreatment suppressed profilin-induced [3H]thymidine incorporation (Figure 3C), confirming that [3H]thymidine incorporation was specifically increased by profilin itself.

Extracellular Profilin Activation of AP-1 DNA Binding in MC
Because the AP-1 transcription factor complex has been implicated as a key element in cell signal transduction in response to stimuli such as growth factors, cytokines, and stress leading to cell growth (38), we examined whether extracellular profilin affected AP-1 DNA-binding activity. Using gel-shift analysis, we observed that profilin increased TRE-binding protein levels in rat MC (Figure 4A). Levels of the TRE-binding complex increased after 1 h, achieved a maximal 2.1-fold increase at 3 h, and returned to basal values at 6 h (Figure 4B). The control nuclear extracts demonstrated no significant changes. A time-course study of AP-1 DNA-binding activity at earlier times (0 to 30 min) revealed no notable changes (data not shown).



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  		Figure 4. Gel-shift analysis of profilin-induced AP-1 DNA-binding activity (A) and time course of splenic profilin-induced AP-1 DNA-binding activity (B). Nuclear extracts were prepared from control or profilin (6.7 µM)-treated cultured rat MC. Equal amounts (5 µg of protein) of the nuclear extracts were assayed for the ability to bind a 32P-labeled 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE) oligonucleotide (A). The positions of AP-1 and the free probe are indicated. The relative amounts of DNA-binding activity are expressed as ratios with respect to the value at time 0 (B). Data are mean ± SD from three independent experiments.

 

Dose Dependence of AP-1 Activation
We also analyzed the dose dependence of profilin-induced AP-1 DNA-binding activity in a gel-shift analysis (Figure 5). Similar to the results obtained in the [3H]thymidine incorporation experiments, profilin induced the activation of AP-1 DNA binding with a threshold of 10 nM; maximal induction was observed at 1 µM. With >100 nM profilin, there was a maximal 2.1-fold increase in AP-1 DNA-binding activity. Therefore, the profilin-induced AP-1 activation was concentration-dependent and saturable (ED50 = 30 nM).



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  		Figure 5. Dose dependence of profilin-induced AP-1 DNA-binding activity. Nuclear extracts were prepared from splenic profilin-treated (logarithmically increased from 1 nM to 10 µM, 3 h) cultured rat MC. Aliquots (5 µg of protein) of the extracts were assayed for the ability to bind the TRE (A). The relative amounts of DNA-binding activity are expressed as ratios with respect to the value of the control (B). Data are the mean ± SD from four independent experiments.

 

Specific Activation of AP-1 by Profilin
We further excluded the possibility that AP-1 induction might have resulted from contamination and, to confirm that profilin specifically activates AP-1, we examined the effects of anti-profilin antibody on profilin-induced activation of AP-1 DNA binding. Before profilin stimulation of MC, profilin was preincubated with anti-profilin antibody. This pretreatment suppressed profilin-induced AP-1 activation (Figure 6A). We also attempted to exclude the possibility that our experimental results might be affected by contaminating splenic growth factors, by using recombinant profilin produced in E. coli. This recombinant profilin also bound to a specific receptor (Figure 2B) and activated c-jun gene expression (Figure 7) and [3H]thymidine incorporation (Figure 3B) as effectively as did splenic profilin. Taken together, these observations indicate that the activation of AP-1 was specifically induced by profilin.



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  		Figure 6. Specific induction of AP-1 by profilin. (A) Suppression of AP-1 activation by anti-profilin antibody. Before a 3-h profilin treatment, splenic profilin (100 nM) was preincubated for 1 h at 37°C in culture medium with or without 1 to 10 µg/ml anti-profilin antibody. Equal amounts (5 µg of protein) of the nuclear extracts were assayed for the ability to bind the 32P-labeled TRE. Data are representative of three independent experiments. (B) Effect of heat denaturation of profilin on AP-1 DNA-binding activity. Purified splenic profilin was boiled for 5 min. MC were incubated for 3 h with phosphate-buffered saline (PBS), 6.7 µM profilin, or 6.7 µM heated profilin. Aliquots (5 µg of protein) of the extracts were assayed for the ability to bind the TRE. Relative DNA-binding activities were 100 ± 6, 199 ± 27, and 106 ± 6, respectively (mean ± SD from three independent experiments; control = 100). (C) Supershift analysis of profilin-induced AP-1. Nuclear extracts (5 µg of protein) prepared from splenic profilin-treated (6.7 µM, 3 h) MC were assayed for the ability to bind the TRE. An antibody specific for c-Jun was included in the gel-shift reaction 2 h before the incubation. As a specificity control, a 100-fold excess of unlabeled probe was added.

 


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  		Figure 7. Competitive reverse transcription (RT)-PCR analysis of MC c-jun gene expression activated by recombinant profilin. (A) Competitive RT-PCR assays after addition of internal standard templates to MC cDNA. Poly(A)+ RNA were purified from MC treated for 1 h with no addition (Control), with an equivalent volume from a sonicate of mock-transformed E. coli (Mock), or with 6.7 µM recombinant profilin purified from a sonicate of E. coli transformed with pRPROF (Profilin). RT-PCR yielded a 402-bp product (arrowhead marking c-jun) that was separated from the 243-bp internal standard bands (arrowhead designated competitor). (B) Competitive PCR quantification of recombinant profilin-induced MC c-jun levels. The control, mock, and recombinant profilin-induced c-jun bands and internal standard bands shown in A were analyzed by calculating the c-jun/ internal standard ratios, using densitometric values; data were plotted after defining the control as 1. Values are mean ± SD from three separate experiments.

 

Profilin that had been boiled for 5 min failed to increase AP-1 DNA-binding activity (Figure 6B), suggesting that the native three-dimensional protein structure of the profilin molecule is required for its activity. To determine whether this profilin-induced TRE-binding complex was an AP-1 complex containing a c-Jun subunit, we performed a supershift analysis with the nuclear extract from rat MC, using an anti-c-Jun antibody. An antibody specific for c-Jun completely upshifted the TRE-binding complex (Figure 6C, lane 5). As a specificity control, a 100-fold excess amount of unlabeled probe was added as a competitor. This control resulted in complete competition for profilin-induced AP-1 DNA binding (Figure 6C, lane 4), indicating that the profilin-induced TRE-binding complex was AP-1 containing c-Jun.

Effects of Profilin on c-jun Gene Expression
AP-1 consists of homo- or heterodimeric protein complexes formed by the related jun and fos gene family members. Therefore, we next examined the effects of profilin on c-jun and c-fos gene expression. mRNA levels in profilin-treated cells were compared with the levels in control cells by competitive RT-PCR. Serial dilutions of internal standard DNA, which generated a band distinct from native bands, were added to equal amounts of reverse-transcribed MC mRNA before amplification. The reaction scheme and corresponding ethidium bromide-stained gels after PCR are shown in Figure 8A. The amount of MC cDNA was calculated from the equivalence point (Y = 1) by linear regression analysis (Figure 8B). The c-jun mRNA levels in control and profilin-treated rat MC were 0.91 and 1.79 amol/250 ng poly(A)+ RNA, respectively. Recombinant profilin also induced c-jun gene expression (Figure 7). Thus, in rat MC, profilin treatment resulted in a 2.0-fold increase in c-jun mRNA levels at 1 h.



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  		Figure 8. Competitive RT-PCR quantification of MC c-jun activated by splenic profilin. (A) Reaction scheme and corresponding ethidium bromide-stained gel after PCR amplification. Competitive RT-PCR assays were performed by adding decreasing amounts of internal standard templates to the MC cDNA. The test templates were reverse-transcribed from control or 6.7 µM profilin-treated MC poly(A)+ RNA for 1 h and yielded a 402-bp PCR product that was electrophoretically separated from the 243-bp internal standard band. (B) Competitive PCR quantification of profilin-induced MC c-jun levels. The test and internal standard bands shown in A were analyzed by plotting the internal standard/wild-type ratios, determined using densitometric values, versus the amount of internal standard cDNA in each tube. The amount of cDNA in the wild-type samples was calculated from the equivalence point (Y = 1).

 

The kinetics of the response are shown in Figure 9 (A and B). The level of c-jun mRNA increased 2.1-fold at 1 h and returned almost to the basal value by 6 h. The time course of c-jun mRNA expression was also studied for short periods (0 to 30 min), and no notable change was detected (data not shown). The levels of c-fos and ß-actin mRNA expression were less affected by profilin (Figure 9, A and B).



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  		Figure 9. Effects of profilin on the expression of c-jun, c-fos, and ß-actin mRNA. (A and B) Time course of c-jun, c-fos, and ß-actin mRNA expression. Cultured rat MC were incubated with 6.7 µM splenic profilin for the indicated times. Equal amounts (250 ng) of poly(A)+ RNA were reverse-transcribed and amplified by PCR with specific PCR primers for c-jun, c-fos, and ß-actin. After gel electrophoresis, the PCR products stained with ethidium bromide (A) were quantified by scanning densitometry. Relative mRNA expression levels are presented as ratios with respect to values at time 0 and are mean ± SD from three independent experiments (B). (C) Effect of cycloheximide on profilin-induced AP-1 activation. Before a 3-h splenic profilin treatment, MC were preincubated for 15 min in medium containing 50 ng/ml cycloheximide. Nuclear extracts (5 µg of protein) were assayed for their ability to bind the TRE. Relative DNA-binding activities were 100 ± 5, 215 ± 25, 105 ± 13, and 110 ± 26 for the extracts in lanes 1 to 4, respectively (mean ± SD from three independent experiments; control = 100).

 

Because profilin-induced AP-1 activation demonstrated a late response, compared with that of growth factors, and was accompanied by c-jun mRNA expression, we next examined whether AP-1 induction required de novo protein synthesis. MC were preincubated with cycloheximide (50 ng/ml, 15 min) before profilin treatment. Cycloheximide completely blocked the profilin-induced AP-1 activation (Figure 9C), suggesting that the AP-1 induction required de novo synthesis of AP-1 components.

Effects of Profilin on PKC Activity
AP-1 can be stimulated directly or indirectly by signaling cascades involving PKC, Ras, Raf-1, and mitogen-activated protein kinase pathways (38). To identify the upstream pathways leading to AP-1 induction by profilin, we examined whether the increase in AP-1 DNA-binding activity in profilintreated cells depended on signaling cascades involving PKC. PKC is activated by its translocation from the cytosol to the cell membrane. In rat MC, 2- to 5-min treatment with 6.7 µM profilin resulted in a rapid and transient increase in PKC activity in the membrane fraction (Figure 10A). The degree of the increase was similar to that observed with TPA stimulation. Decreased PKC activity was observed in the cytosolic fraction in parallel with the increase in the activity of the membrane fraction (control, 1199.7 ± 26.7 pmol 32P incorporated/min per mg protein; profilin, 821.2 ± 56.9 pmol 32P incorporated/min per mg protein; TPA, 790.0 ± 92.7 pmol 32P incorporated/min per mg protein; mean ± SD from three experiments performed in duplicate; P < 0.01 for profilin and TPA values versus control values). The level of cAMP in cultured rat MC was also measured by RIA, as described (39). No change in the cAMP concentration was observed after a 10-min incubation with 6.7 µM profilin (control, 334.0 ± 5.3 pmol/mg protein; profilin, 320.0 ± 12.5 pmol/mg protein; mean ± SD from four separate experiments). We also tested the effects of two PKC inhibitors, H-7 and staurosporine, on profilin-stimulated AP-1 DNA-binding activity. Both H-7 and staurosporine blocked profilin-induced AP-1 DNA binding to nearly control levels (Figure 10B, lanes 3 and 4), further suggesting that the profilin induction of AP-1 is PKC-dependent.



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  		Figure 10. Effects of profilin on protein kinase C (PKC) activity. (A) Effects of profilin and TPA on PKC activity in cultured rat MC. The cells were incubated for the indicated times in PBS containing either 6.7 µM splenic profilin, 200 nM TPA, or PBS alone. PKC activity in the particulate fractions was expressed as picomoles of 32P incorporated per minute per milligram of protein (mean ± SD) from three duplicate experiments ({dagger}P < 0.01 versus control). (B) Effects of PKC inhibitors on profilin-induced AP-1 binding activity. Before a 3-h treatment with 6.7 µM splenic profilin, MC were preincubated for 2 h with or without 10 µM H7 or 10 nM staurosporine. Aliquots (5 µg of protein) of the extracts were assayed for their ability to bind the TRE. Relative DNA-binding activities were calculated from three independent experiments (control = 100).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated, using classic Scatchard analysis, the existence of a novel, saturable, high-affinity receptor mechanism for profilin binding to MC surfaces. A single class of high-affinity receptors (Kd = 30 to 60 nM) could be identified using both profilin isolated from spleen and recombinant profilin. The equivalent activity of purified recombinant profilin rules out cell surface binding via trace contamination with any cytoskeletal protein. At least three intracellular molecules that bind to profilin have been previously reported, actin, PIP2, and VASP (3,4,5). To avoid contamination by intracellular molecules that may bind to profilin during binding experiments, we used intact MC rather than isolated membrane fractions. PIP2 and VASP are unlikely ligands for profilin on the cell surface, because of their intracellular distribution. Although actin (a major profilin-binding protein) is primarily an intracellular constituent, it has also been identified on the surface of several cell types (40). However, the binding properties of actin binding to profilin (Kd = 1 to 10 µM) (41,42) are quite different from the high-affinity binding of profilin to MC. Therefore, it is unlikely that the binding site for profilin on MC is actin; rather, our findings suggest the existence of a novel receptor for profilin. The molecular identity of the cell surface receptor for profilin remains to be determined.

We demonstrated a concentration-dependent induction of AP-1 DNA-binding activity by profilin, with an ED50 of 30 nM. That concentration was very similar to the Kd value for profilin binding. These findings indicate that profilin increases AP-1 DNA-binding activity through a specific high-affinity receptor on rat MC. This study is the first report that extracellular profilin can activate intracellular signaling through binding to a specific cell surface receptor.

We also found that profilin induced a time-dependent increase in AP-1 DNA-binding activity in rat MC. The AP-1 transcription factor is a downstream nuclear effector in signal transduction cascades stimulated by the activation of various growth factor receptors. The activation of AP-1 is regulated by complex mechanisms that involve distinct modifications of the constitutively existing AP-1 complex by phosphorylation or de novo synthesis of an AP-1 subunit. Mitogenic stimulation, e.g., by TPA or ultraviolet light, induces phosphorylation of the amino-terminus of c-Jun (on serine-63/73) within minutes; this is responsible for the transactivation activity of c-Jun and does not require new protein synthesis for AP-1 induction (43). c-Jun is also dephosphorylated at the carboxyl-terminal DNA-binding domain in response to cell stimulation; within minutes, an increase in DNA-binding activity is observed (44). Our study demonstrated that profilin produced an increase in c-jun mRNA levels at 1 to 3 h, followed by an increase in AP-1 DNA-binding activity at 3 h. Therefore, in our time-course study, c-jun gene expression stimulated by profilin paralleled but preceded the induction of AP-1. We also found no significant change in c-jun mRNA expression or AP-1 DNA-binding activity when exposure to profilin was of short duration (0 to 30 min). Furthermore, cycloheximide completely blocked profilin induction of AP-1. These results suggest that AP-1 activation by profilin may be mediated by de novo synthesis of the c-Jun AP-1 subunit. Recombinant profilin produced in E. coli demonstrated the same effects as splenic profilin on c-jun gene expression. This finding further supports the concept that extracellular profilin can induce signal transduction.

AP-1 is activated in response to many growth factors and other agents, and it controls cell growth, apoptosis (45), and the transcription of TRE-containing genes, e.g., transforming growth factor-ß (TGF-ß) (46), endothelin-1 (47), insulin-like growth factor II (48), pro-{alpha}1(I) collagen (49), collagenase type I (18), and c-jun (50), which are also implicated in the progression of renal disease. Because profilin significantly activated AP-1, we examined its effects on the growth of rat MC in culture. In this study, [3H]thymidine incorporation was increased 1.9-fold, indicating that profilin can act as a mitogen. Although profilin was not as potent as PDGF, stimulation of cells with growth factors together with profilin demonstrated additive effects on [3H]thymidine incorporation. These findings suggest that the signal transduction by profilin may be relevant to MC pathologic processes, in cooperation with growth factors, in glomerular diseases. It might be interesting to investigate whether profilin-induced activation of AP-1 can affect the expression of TRE-containing genes such as TGF-ß, because TGF-ß is a key factor that is responsible for the progression of glomerular sclerosis in many types of glomerulonephritis (13).

Our data also demonstrated that extracellular profilin induced rapid translocation of PKC from the soluble fraction to the membrane fraction of MC. The magnitude of the increased membrane PKC activity produced by profilin was similar to that observed with phorbol ester stimulation. To investigate the upstream pathway leading to AP-1 activation, we examined the effects of two PKC inhibitors on profilin-induced AP-1 activation. Both staurosporine and H-7 diminished AP-1 activation nearly to control levels. These findings suggest that profilin induces AP-1 activation via a PKC-dependent pathway. However, the time lag between AP-1 and PKC activation (1 to 3 h for AP-1 versus minutes for PKC) suggests that several signaling molecules are involved. The failure of heat-denatured profilin to activate AP-1 suggests that the native three-dimensional structure of the molecule is required for its interaction with the receptor, as is also observed for the lower-affinity association of profilin with actin (37).

Profilin produced an increase in TRE-binding protein levels in rat MC. Results from our supershift analysis using an anti-c-Jun antibody and our competition study with excess unlabeled TRE probe led us to conclude that the increased TRE-binding complex stimulated by profilin is an AP-1 complex containing a c-Jun component. The inhibition study with neutralizing antibody (anti-profilin peptide antibody) confirmed that the reaction was specifically induced by profilin and was not the result of contamination by any growth factor (from insufficient purification). The study also suggested that the phenylalanine-84 to serine-92 sequence in profilin may play an important role in the profilin-receptor interaction. Recombinant profilin also demonstrated specific high-affinity binding and induction of c-jun gene activation, thus ruling out the possibility that some splenic growth factor or other molecule contaminating the preparations could account for the profilin effects.

In conclusion, we have established the existence of a specific cell surface receptor for profilin and have presented findings regarding signal transduction functions of profilin in rat glomerular MC. Profilin, which is released into the extracellular space in some pathologic situations, can stimulate AP-1 activity and DNA synthesis in vitro. These findings suggest a novel signaling function for profilin and implicate profilin in some glomerular diseases.


   Acknowledgments
 
We thank Drs. Ira Pastan (National Institutes of Health, Bethesda, MD) and Sunao Fujimoto (University of Occupational and Environmental Health, Kitakyushu, Japan) for their useful advice during this study. We also thank Drs. Jianguo Gu (Osaka University, Osaka, Japan), Kazue Matsumoto (National Institutes of Health), and Akira Yashiro, Yoshiaki Doi, Hideaki Kudo, and Akiko Sugimoto (University of Occupational and Environmental Health) for their assistance. This work was supported by research grants from the University of Occupational and Environmental Health (HH), the Fukuoka Cancer Society (Japan) (MT), the Ministry of Education, Science, and Culture of Japan (Grant 11671065) (AO), and the Renal Anemia Foundation (Japan) (AO, MT).


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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Received for publication November 24, 1999. Accepted for publication February 2, 2000.




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