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Platelet-Derived Growth Factor-BB Stimulates Hypertrophy of Peritubular Smooth Muscle Cells from Rat Testis in Primary Cultures¹

Department of Histology and Medical Embryology and Centro Acidi Nucleici (E.B.), Consiglio Nazionale delle Ricerche, La Sapienza University, 00161 Rome, Italy

Address all correspondence and requests for reprints to: Dr. Fioretta Palombi, Department of Histology and Medical Embryology, Via A. Scarpa 14; 00161 Rome, Italy. E-mail: fioretta.palombi{at}uniroma1.it

	Abstract

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The tunica propria of seminiferous tubules contains a particular type of smooth muscle cell (myoid cells) arranged in a contractile epithelioid layer that is responsible for sperm and tubular fluid flow. Unlike other types of smooth muscle (SM) cells, highly purified populations of peritubular smooth muscle cells (PSMC) survive and maintain their contractile phenotype in primary cultures in controlled conditions. We used this culture model to investigate the response of the SM contractile phenotype to prolonged exposure to platelet-derived growth factor (PDGF), one of the main factors involved in vascular SM pathologies. We observed that 4-day continuous exposure of PSMC to PDGF-BB at nanomolar concentrations in plain medium enhances contractile phenotype traits and induces cell hypertrophy without inducing proliferation. In Northern and Western blotting experiments, SM--actin transcript and protein were found to be markedly increased in the PDGF-BB-treated samples, which is in line with the formation of conspicuous SM--actin-containing stress fibers. Moreover, binding sites for endothelin-1 were increased, and the calcium response to the contractile agonist, determined in single fura-2-loaded cells, was enhanced. In response to PDGF-BB, the cells underwent immediate, transient contraction, as seen in a scanning electron microscope, followed by a gradual increase in size, as evaluated by cytofluorometry, and enhancement of protein synthesis. The observed pattern of response to PDGF-BB was not accompanied by cell proliferation, as assessed by [³H]thymidine incorporation and direct cell counts. Unlike other SM cell types, in which proliferation and loss of contractile traits are induced by PDGF, chronic treatment of PSMC with this growth factor results in hypertrophy rather than hyperplasia.

	Introduction

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THE RAPID progression of spermatozoa along the lumen of seminiferous tubules toward the hilum of the testis depends upon the functional activity of peritubular smooth muscle cells (PSMC) (1), contractile elements arranged in a continuous sheath around the seminiferous epithelium and traditionally referred to as myoid cells. In recent years experimental data have focused on the peculiarities of these cells within the heterogeneous smooth muscle family and on the paracrine regulation of their contractile function. PSMC express desmin (2), smooth muscle specific myosin (3), and -actin (SM--actin) (4, 5); although they display the ultrastructural characteristics of smooth muscle cells (6), they are arranged in a flat epithelioid layer, which forms the peculiar tunica propria of the seminiferous tubule, and contribute to the blood-testis permeability barrier (7, 8, 9). In the apparent absence of nerve endings, peritubular contractions are controlled by local factors released by the adjacent compartments, interstitium and seminiferous epithelium. Recently, in vitro and ex vivo studies in the rat have shown that the local factors endothelin (10, 11), vasopressin (12), and PGF₂ (13), are capable of stimulating PSMC contraction. In particular, we have shown that in the adult rat the production of biologically active endothelin by Sertoli cells is subject to spatial and temporal control according to seminiferous epithelium cyclicity (14). Additional interaction between PSMC and the seminiferous epithelium is represented by the cooperative production of basal lamina components (15). This is a point of particular interest, as tubular matrix overaccumulation (termed hyalinization) is a feature shared by primary fertility disorders as diverse as cryptorchidism, Klinefelter’s syndrome, and orchitis, among others (16). In vascular smooth muscle cells (VSMC), a cell type with many similarities to PSMC, matrix overproduction is known to be related to loss of phenotypic traits; phenotype transition from the contractile to synthetic and secretory states is regarded as a key early event in the pathogenesis of atherosclerosis and in arterial injury response (17, 18). Although the possible signals that directly or indirectly stimulate overproduction of the peritubular matrix in pathological conditions and/or affect the contractile phenotype of PSMC are as yet unknown, the expression of matrix components is known to be stimulated in vitro in normal PSMC by platelet-derived growth factor (PDGF) (19), a factor responsible for loss of the contractile phenotype in VSMC pathology (20). As PDGF is produced in the testis by both interstitial Leydig cells (21) and Sertoli cells (22) and has been implicated in the paracrine regulation of peritubular matrix production (19), we have analyzed the response of rat PSMC to prolonged exposure to the factor in vitro with the aim of elucidating whether their contractile phenotype is affected by a local factor known to up-regulate matrix biosynthesis. The results obtained show that, unlike their vascular counterparts, the response of PSMC to PDGF consists in an increase in size, potentiation of their contractile apparatus, and increased sensitivity to endothelin, but not in cell proliferation. This unexpected response to PDGF indicates that PSMC primary cultures represent a promising new model in which the mechanisms of smooth muscle hypertrophy can be investigated in unique, controlled conditions.

	Materials and Methods

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Animals
The animals used were 18- to 20-day-old Wistar rats (Charles River Laboratories, Inc., Como, Italy). Animals were kept in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were killed by CO₂ asphyxia. Eight to 10 rats were used for each experiment.

Cell isolation, culture, and treatments
Purified myoid cells were prepared as previously described (10, 23). Briefly, the peritubulum was detached from trypsin-dispersed seminiferous tubules by collagenase digestion and subjected to centrifugation at 40 x g. The pellet was further digested in trypsin and EDTA to a single cell suspension and fractionated on a discontinuous Percoll density gradient. Collagenase A from Clostridium histoliticum and deoxyribonuclease I were obtained from Roche Molecular Biochemicals (Mannheim, Germany), trypsin was purchased from Difco Laboratories (Detroit, MI), and Percoll was obtained from Pharmacia Biotech (Uppsala, Sweden). The cells were routinely seeded on Falcon plastic dishes (Becton Dickinson and Co., Franklin Lakes, NJ), except where otherwise specified, at a concentration of 50 x 10³ cells/cm² and were cultured under serum-free conditions at 37 C in a humidified atmosphere of 5% CO₂ and 95% air in Eagle’s MEM (Life Technologies, Inc., Grand Island, NY). Cell purity, assessed for each preparation by alkaline phosphatase cytochemistry (24), was never below 95%. Stimulation with human recombinant PDGF-BB (from Roche Molecular Biochemicals) at a concentration of 50 ng/ml was initiated after 2 days of culture in plain medium and continued for the times indicated. In one experiment the treatment was initiated after 4 days to verify that the cells were able to respond to delayed PDGF stimulation. In the experiments aimed at verifying the specificity of the response to the growth factor, the PDGF receptor tyrosine kinase activity inhibitor CGP53716 (25) (a gift from Dr. E. Buchdunger, Novartis Pharma, Basel, Switzerland) was used at a 10-µM concentration. In these last experiments, treatment with the inhibitor was initiated 1.5 h before the addition of PDGF-BB. In an experiment designed to test whether the morphological response to PDGF was reversible, the growth factor was withdrawn after 24–96 h of stimulation, and 10 µM CGP53716 was added for up to 4 additional days. The medium was changed every 2 days in all of the experiments.

Scanning and transmission electron microscopy
For scanning electron microscopy, the cells were fixed in 2.5% buffered glutaraldehyde 2 min after stimulation and postfixed in OsO₄. Before dehydration and critical point-drying in ethanol, the dishes were carefully cut into small fragments by means of a sharp blade. The samples were coated with gold and viewed in a Hitachi S-570 scanning electron microscope (Milan, Italy). The ultrastructural modifications induced by 4-day PDGF treatment were studied after cells had been detached by 10-min incubation in 0.02% EDTA followed by 3-min digestion in 0.05% trypsin-0.02% EDTA (HyClone Laboratories, Inc., Cramlington, NE). The samples were fixed in 2.5% buffered glutaraldehyde, postfixed in OsO₄, dehydrated in ethanol, embedded in epoxy resin, and viewed in a Hitachi 7000 transmission electron microscope.

SM--actin detection by immunofluorescence
Cells were fixed in cold ethanol/acetone (1:1, vol/vol) for 10 min and pretreated with 2% BSA before incubation with monoclonal anti--smooth muscle actin (1:300; clone 1A4, Sigma, St. Louis, MO); the secondary antibody used was antimouse fluorescein isothiocyanate-conjugated secondary antibody, absorbed with rat serum proteins (also from Sigma). The specificity of immunolabeling was verified in control samples prepared with the primary antibody omitted.

Proliferation
To evaluate a possible increase in cell number after PDGF treatment, direct evaluation of cell density per unit area was performed on acetone/ethanol-fixed cells after nuclear staining with Hoechst 33342 (Sigma). Random photographic fields were obtained with a fluorescence microscope and subjected to nuclear counts. In each experiment, 2,500 nuclei were scored for each experimental condition. For [³H]thymidine incorporation analysis, cells were cultured in 12-multiwell plates (150,000 cells/well) and exposed to 50 ng/ml PDGF-BB or 10% FCS (Life Technologies, Inc.) for 1–3 days. The cells were labeled with 1 µCi/ml [methyl-³H]thymidine (SA, 20 Ci/mmol; NEN Life Science Products, Boston, MA) for an additional 18 h in the same culture conditions. The radioactivity incorporated into DNA was evaluated after trichloroacetic acid precipitation of macromolecules and measured by liquid scintillation spectrometry.

Amino acid incorporation
Both unstimulated cells and those stimulated with PDGF for 24–96 h were labeled with 10 µCi/ml [³⁵S]methionine (SA, 1175 Ci/mmol; NEN Life Science Products) for 2 h in MEM without methionine. Cells were lysed in 50 mM Tris, 100 mM EDTA, 100 mM NaCl, and 0.1% SDS, and the lysate was split in equal amounts for DNA extraction and radioactivity incorporation determination. For DNA extraction, the lysate was incubated for 2 h in the presence of 0.5 mg/ml proteinase K (Sigma) at 55 C. After digestion, DNA was extracted with phenol-chloroform-isoamyl alcohol. The pellet was resuspended in 10 mM Tris-HCl and 1 mM EDTA, pH 8, and the DNA content was determined by a fluorometric assay using Hoechst 33258 (Sigma) as the fluorescent dye. An aliquot (2 µl) of the sample was added to 2 ml dye solution (0.1 µg/ml Hoechst in 0.1 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Fluorescence was immediately measured by a Perkin-Elmer Corp. fluorometer (Norwalk, CT) at 365/460 nm (excitation/emission) wavelengths, using salmon sperm DNA as a standard. To evaluate [³⁵S]methionine incorporation, the parallel lysate was subjected to trichloroacetic acid precipitation, and the radioactivity incorporated into proteins was measured by liquid scintillation spectrometry. The radioactivity was normalized to DNA (determined as described above).

Measurement of intracellular Ca²⁺([Ca²⁺]_i)
Measurement of [Ca²⁺]_i was performed on cells plated onto plastic coverslips as previously described (10, 14). Briefly, [Ca²⁺]_i was measured by dual wavelength fluorescence in single cells loaded with the Ca²⁺-sensitive indicator fura-2. Samples pretreated for 4 days with PDGF and parallel controls were incubated in 1 ml serum-free MEM containing 3 µM fura-2-acetoxymethylester for 1 h at 37 C. The cells were then rinsed with Krebs-Henseleit-HEPES buffer (140.7 mM Na⁺, 5.3 mM K⁺, 132.4 mM Cl^-, 0.98 mM PO₄^2-, 1.25 mM Ca²⁺, 0.81 mM Mg²⁺, 5.5 mM glucose, and 20.3 mM HEPES) supplemented with 0.2% fatty acid-free BSA. [Ca²⁺]_i-dependent fluorescence was measured in single cells, at 340- and 380-nm excitation wavelengths, before and after addition of 10 nM endothelin-1 (ET-1; Peninsula Laboratories, Inc., Belmont, CA) with a microfluorometer (SPEX Industries, Edison, NJ) connected to a Nikon (Sesto Fiorentino, Italy) Diaphot-TMD inverted microscope equipped with a Nikon CF x40 fluor objective. Emission was collected by a photomultiplier carrying a 510-nm cut-off filter and was recorded by an ASEM Desk 2010 computer that automatically calculated real-time 340/380 ratios. Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca²⁺-dependent fura-2 fluorescence with 5 µM of the Ca²⁺ ionophore ionomycin, followed by a recording of the minimal fluorescence after the addition of 7.5 mM EGTA and 60 mM Tris-HCl, pH 10.5. [Ca²⁺]_i was calculated using previously described formulas (26).

[¹²⁵I]ET-1 binding
To analyze binding sites for ET-1, binding competition experiments were performed on cells cultured in 24-multiwell plates (150,000 cells/well) treated or not treated with PDGF for 4 days. Cells were rinsed in MEM containing 0.2% BSA, then incubated in 170 µl of the same medium. Nonradioactive endothelin at graded concentrations of 50 pM to 1 µM was added, in 20-µl aliquots, to evaluate the ability to compete with the radioligand; [¹²⁵I]ET-1 (SA, 2,200 Ci/mmol; NEN Life Science Products) was added, in 10-µl aliquots, to a final concentration of 50 pM, and incubation was performed for 90 min at 37 C. The cell-bound radioactivity was measured after removal of the incubation mixture and washing of the cells with cold MEM-0.2% BSA. The remaining cell-associated radioactivity was quantitated after solubilization of cells in 1 M NaOH. Nonspecific binding, determined by a parallel incubation in the presence of unlabeled ET-1 (1 µM), never exceeded 2% of the total cell-associated radioactivity. All data points were measured in triplicate.

Flow cytometry
Cells stimulated with PDGF for 24–96 h and parallel control samples were detached by 10-min incubation in 0.02% EDTA followed by 3-min digestion in 0.05% trypsin-0.02% EDTA, and finally resuspended in 0.1% BSA in HBSS. Changes in cell size [forward scatter (FS)] and complexity [side scatter (SS)] were studied by means of a Coulter EPICS XL cytometer (Beckman Coulter, Inc., Fullerton, CA). Cells were gated using FS vs. SS to exclude cell debris and aggregates. For each condition 5000 events were acquired in the linear mode.

Immunoblotting
The cells were lysed and scraped in a commercial cell lysis buffer (New England Biolabs, Inc., Beverly, MA) containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na₃VO₄, and 1 µg/ml leupeptin, to which 2% SDS and 1 mM phenylmethylsulfonylfluoride were added. Control and treated samples were lysed in equal volumes of lysis buffer. The protein concentration of each sample was determined using the microbicinchoninic acid method (Pierce Chemical Co., Rockford, IL). Equal volumes of cell lysate (50 µl) or proteins (30 µg) were run on a 12% SDS-polyacrylamide gel in reducing conditions, followed by transfer onto a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech, Aylesbury, UK). The blots were saturated with 5% BSA (Sigma) and probed with 1:2500 monoclonal anti-SM--actin (clone 1A4, Sigma). The secondary antibody used was horseradish peroxidase-conjugated rabbit antimouse antiserum (Zymed Laboratories, Inc., South San Francisco, CA), and detection was performed using the chemiluminescence system (ECL, Amersham Pharmacia Biotech). Bands were quantitated using an LKB Ultrascan XL laser densitometer (Pharmacia Biotech, Uppsala, Sweden).

RNA extraction and Northern blotting
Total RNA was extracted from 12 x 10⁶ cells with RNAzol B (Biotecx, Inc., Houston, TX). Equal amounts of total RNA (12 µg/lane) were electrophoresed on 0.8% agarose-formaldehyde gels (27) at 4 C and capillary-transferred overnight onto Hybond-N membranes (Amersham Pharmacia Biotech). The blots were prehybridized for 6 h at 37 C in 50% formamide, 6 x SSC, 5 x Denhardt’s solution, and 0.1% SDS and hybridized overnight in the same buffer with a 28-mer synthetic oligonucleotide with the sequence 5'-CATTCACAGTTGTGTGCTAGAGACAGAG-3', complementary to the 3'-untranslated region of the rat SM--actin messenger RNA (mRNA; accession no. M22757). The deoxynucleotide had been labeled at the 5'-end with [-³²P]deoxy-ATP and polynucleotide kinase (Roche Molecular Biochemicals), according to the method of Sambrook et al. (27). As a control for RNA integrity and quantitation, the blots were stripped of the SM- actin probe and hybridized in the same conditions with a 5'-end ³²P-labeled 21-mer oligonucleotide, 5'-AACCATAACTGATTTAATGAG-3', complementary to the 5'-region of the rat 18S ribosomal RNA (rRNA; accession no. V01270). Counts were quantitated with a Packard A2024 Instantimager (Downers Grove, IL).

Statistical analysis
Student’s t test was used for statistical comparison between means where applicable. Binding data were subjected to Scatchard analysis. Linear least square analysis of the Scatchard plot was performed with the aid of the computer program BDATA-EMF (28).

	Results

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Shape changes and remodeling of the contractile apparatus in response to PDGF-BB
The morphological response to PDGF was studied both immediately after stimulation and after 1–4 days of continuous treatment. In the scanning electron microscope, at all times during the first week of culture, the cells in plain medium appeared nonoriented and flat; the only bulging profile corresponded to the nuclear area. The laminar cytoplasm had a regular outline, with the exception of occasional protrusions (Fig. 1a). Cell contraction occurred within 2 min of PDGF addition and resulted in pronounced bulging of most of the cell body accompanied by multiple blebbing, with cellular attachment to the substrate apparently maintained through numerous slender processes (Fig. 1b). This acute response was transient and lasted about 10–15 min (not shown). With continued exposure to the factor, the cells acquired an oriented shape, which was first apparent in the light microscope after 1 day (not shown). A gradual progress of elongation and distension took place from day 2 to 4, which resulted in a fibroblast-like morphology (Fig. 1, c and d) and acquisition of an oblong nuclear shape (Fig. 3). The presence and distribution of SM--actin were studied to evaluate whether prolonged PDGF treatment alters the PSMC contractile phenotype. In control medium, slender SM--actin-containing filaments appeared mainly radially oriented, reflecting the unoriented cell shape. This pattern remained unchanged for at least 6 days (Fig. 2a). In PDGF-treated samples, conspicuous SM-actin-positive stress fibers appeared 24 h after stimulation (not shown) and continued to develop upon continuation of the treatment for up at least 4 days (Fig. 2b). A comparable pattern of response to PDGF was observed when treatment was initiated after 4 days of incubation in plain medium and continued for the following 48 h (Fig. 2c), demonstrating that in our control samples sensitivity to the factor is maintained for days. The same morphological changes and actin filament reorganization were observed in preliminary experiments, although to a minor degree, at a PDGF concentration as low as 10 ng/ml (not shown). In all cases the PDGF response was inhibited (Fig. 2d) in the presence of 10 µM CGP53716, an inhibitor of PDGF receptor tyrosine kinase activity that does not affect either morphology or actin filament distribution in parallel control samples (not shown). In experiments aimed at evaluating the possible reversibility of the response, PDGF was withdrawn, and the inhibitor added at different time intervals after stimulation; in these conditions, the morphological response was arrested, but failed to revert to the condition of unstimulated samples (not shown).

View larger version (149K): [in this window] [in a new window]   Figure 1. Morphological response of PSMC to acute and prolonged treatment with PDGF-BB. a and b, scanning electron microscopy; c and d, phase contrast. a, Cell morphology in plain medium; b, shape changes observed 2 min after addition of PDGF, on day 2 of culture; c, cell morphology after culture for 6 days in plain medium; d, elongation and spreading in cells precultured in plain medium for 48 h and subsequently stimulated with PDGF for 4 additional days. Magnification: a and b, x4500 (bar, 2 µm); c and d, x150 (bar, 65 µm).

View larger version (53K): [in this window] [in a new window]   Figure 3. Nuclear morphology, but not density/unit area, is modified by prolonged PDGF-BB treatment. a, Cells cultured for 6 days in plain medium, viewed in a fluorescence microscope after Hoechst nuclear staining; b, cells stimulated with PDGF from day 2 to 6 of culture. Nuclear elongation in response to the growth factor is evidenced by Hoechst stain. Magnification: a and b, x320. Bar, 30 µm. c, Equivalent density of cell nuclei/unit area in treated and control cells. Nuclear counts are based on random photographic fields of Hoechst-stained cells. The values shown are the mean of three independent experiments in which over 2500 nuclei were scored for each experimental condition; counts from control and treated samples did not differ significantly (P = 0.8).

View larger version (114K): [in this window] [in a new window]   Figure 2. Distribution of SM--actin-containing filaments in control and PDGF-BB-stimulated samples. a, Cells cultured for 6 days in plain medium. Fluorescence is mainly distributed along radial filaments; b, in response to 4-day treatment with PDGF (from day 2 to 6), well defined SM--actin-containing stress fibers have formed; c, SM--actin-containing stress fibers form even when PDGF treatment is initiated after 4-day culture in plain medium and continued for an additional 48 h; d, cells stimulated with PDGF as described in b, but in the presence of 10 µM CGP53716, an inhibitor of PDGF receptor tyrosine kinase activity. Magnification, x350. Bar, 25 µm.

View larger version (32K): [in this window] [in a new window]   Figure 4. Lack of proliferation in PDGF-BB-stimulated samples. After 1- to 3-day exposure to PDGF or 10% FCS, cells were labeled with [3H]thymidine, and precursor incorporation was evaluated after an additional 18 h in the same culture conditions. Increasing precursor incorporation is apparent in samples treated with FCS, but not with PDGF. The data are representative of three independent experiments.

View larger version (44K): [in this window] [in a new window]   Figure 5. Progressive increase in protein synthesis in response to PDGF-BB. Incorporation of [35S]methionine was evaluated in a 2-h pulse after culturing cells in the presence or absence of PDGF for the indicated times. For DNA extraction and content determination, see Materials and Methods. The data are representative of three independent experiments.

View larger version (33K): [in this window] [in a new window]   Figure 6. Progressive increase in cell size and complexity during prolonged treatment with PDGF-BB. Cultures were treated with PDGF for up to 4 days and subjected to cytofluorometric analysis of FS (upper panels) and SS (lower panels). CTR, Cells cultured in plain medium throughout the experiment; P, cells treated with PDGF for the length of time indicated; relative increases in FS (upper right panel) and SS (lower right panel) at each culture time are shown. The data are representative of three independent experiments and were obtained by analyzing 5000 cells (events) for each condition in each experiment.

View larger version (112K): [in this window] [in a new window]   Figure 7. Ultrastructural changes induced by 4-day stimulation with PDGF-BB. In control (a) as well as treated (b) samples, the cytoplasm appears rich in areas filled with filamentous structures (arrowheads) lined by dense bodies (arrows), shown at higher magnification in the two insets. b, Well developed rough endoplasmic reticulum is only apparent in the PDGF-treated samples. Cells were fixed and processed for transmission electron microscopy after enzyme detachment from the culture dish. Magnification: a and b, x16,000; insets, x32,000. Bar: a and b, 1 µm; insets, 0.5 µm.

Up-regulation of SM--actin synthesis after prolonged PDGF treatment

View larger version (38K): [in this window] [in a new window]   Figure 8. Increase in SM--actin protein and mRNA after PDGF-BB stimulation. Immunoblot determination of SM--actin (left panels) was performed after the cells had been cultured for 4 days in the continuous presence or absence of PDGF. As a hypertrophic response to the factor was expected, control and treated samples were lysed in equal volumes of lysis buffer, and equal amounts of both cells (50 µl cell lysate) and proteins (30 µg) were assayed, as indicated. Densitometric analysis of the immunoblot shows that PDGF treatment induced an increase averaging 40% or 100% when equal amounts of proteins or cell lysate, respectively, were assayed. The data are representative of three independent experiments. Northern blot analysis (right panels) was performed with 12 µg total RNA extracted from cells cultured for 4 days in the continuous presence or absence of PDGF. The probe was a 28-mer deoxynucleotide complementary to the 3'-untranslated region of the rat SM--actin mRNA. A major band of hybridization was detected close to the position of 18S rRNA; when normalized for 18S rRNA, a 6-fold increase in SM--actin mRNA was detected in total RNA of treated cells. As cells cultured in the presence of PDGF contain 5 times more total RNA than control cells, mRNA specific for SM--actin reaches a 30-fold increase in treated cells. The data are representative of two independent experiments.

View larger version (22K): [in this window] [in a new window]   Figure 9. Responsiveness to ET-1 is enhanced in PDGF-BB-treated cells. a, Intracellular calcium mobilization in response to 10 nM ET-1 in fura-2-loaded cells that had been precultured for 4 days in presence or absence of PDGF. The increase in the intracellular calcium concentration ([[Ca2+]) elicited by ET-1 is twice as high in PDGF-pretreated cells as in untreated samples (P < 0.01). The data represent the mean ± SEM of nine determinations obtained from three independent experiments. b, The number of binding sites for ET-1 is increased by prolonged treatment with PDGF-BB. Scatchard analysis of data obtained in binding competition experiments on cells cultured in the absence or presence of PDGF for 4 days. Binding sites average 190,000/cell in control samples and 400,000/cell in PDGF-stimulated samples. The data are representative of two independent experiments.

	Discussion

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Cell proliferation is induced by serum, but not by PDGF
PSMC can be cultured in plain medium in the total absence of serum in nonproliferating conditions and maintain viability and phenotypic characteristics for days. Amino acid incorporation remained constant during the first week of culture, and cell shape as well as the pattern of -isoactin-containing filaments and ultrastructural traits of the SM phenotype appeared to be conserved. In these primary cultures, addition of serum induced cell proliferation accompanied by a progressive increase in [³H]thymidine incorporation. In the same experiments, when stimulated with 50 ng/ml PDGF-BB, the cells failed both to proliferate and to significantly incorporate [³H]thymidine. A previous work (19) reported PSMC proliferation in response to PDGF-BB (at the same concentration and conditions we used), as inferred from an increase in the number of cells recovered after enzymatic detachment from the dish. In our experience this procedure may be misleading and does not represent conclusive evidence of cell proliferation. The proliferation of VSMC in vitro is known to be induced by PDGF as well as by a number of growth factors (17), whereas no single factor or combination of factors has been shown to induce postnatal peritubular cell proliferation to date. Accordingly, in vivo these cells represent a very stable population (8); moreover, unlike VSMC, no peritubular alteration has to our knowledge been demonstrated to involve PSMC hyperplasia.

PDGF treatment results in enhancement of the SM phenotype
An additional unexpected response of PSMC to prolonged PDGF-BB treatment was the increase in -isoactin transcript and protein, which was, on the average, 30- and 2-fold, respectively, compared with that in an equal amount of cells. In SMC, PDGF is known to induce down-regulation of SM--actin mRNA and protein (29), partly through mRNA destabilization (30, 31), as well as of other specific markers of the differentiated state, such as myosin heavy chain and -tropomyosin (32). In this respect, PDGF has been indicated as unique among the factors capable of stimulating SMC proliferation, and its role in the control of the SMC differentiated phenotype has been suggested to be independent of its mitogenic effect (20, 33). The study of intracellular pathways mediating loss or gain of differentiated traits induced by growth factors in SMC has been approached in recent reports. In transfection studies PDGF-BB has been found to induce SMC dedifferentiation through activation of mitogen-activated protein kinase, but to stimulate an increase in differentiation markers when forced through the phosphoinositide 3-kinase/protein kinase B (Akt) pathway (34, 35). It would be interesting to investigate which particular balance in intracellular pathways is responsible for the unexpected increase in SM--actin stimulated by PDGF-BB in testicular PSMC. In our stimulated cells, SM--actin-containing filaments are organized in stress fibers, and the cells appear oriented. On speculative grounds, this very apparent change from the symmetrical shape and radial pattern of actin filaments of control cells might represent an aspect of locomotory behavior, in keeping with the known chemotactic response to PDGF demonstrated for VSMC (36) and perinatal PSMC (37). A further contractile trait potentiated in PSMC by PDGF-BB is the responsiveness to ET-1, evaluated as [Ca²⁺]_i increase in single adherent cells. The response to acute administration of this agonist was significantly higher in cells that had been pretreated for 4 days with PDGF-BB. Although several different causes may underlie this phenomenon, it is interesting to note that the number of binding sites for ET-1 was 100% higher in PDGF-BB-treated samples. To our knowledge, PSMC are the first cell type in which PDGF has been shown to increase responsiveness to ET. These data together with the increase in -isoactin suggest that important traits of the contractile phenotype are potentiated by continued exposure of PSMC to PDGF-BB. When cultured in vitro, SMC are known to undergo phenotypic modulation, a transition in which characteristics of the contractile phenotype are lost and the cells shift to a synthetic phenotype, characterized by an enrichment in rough endoplasmic reticulum and Golgi apparatus and a reduction in myofilaments (38). This modulation is also known to occur in vivo as a response to vascular injury; in this case the modulated cells acquire the ability to colonize the intima, proliferate, and secrete extracellular matrix components (reviewed in Ref. 17 ; 18). PDGF is regarded as one of the main candidates responsible for SMC modulation and, hence, as involved in atherosclerotic intimal thickening (20). The different response of PSMC to this factor indicates that, in this particular type of SMC, the stimulation of matrix hyperproduction by PDGF (19) is not accompanied by a definite loss of contractile phenotype and cannot therefore be regarded as a classic modulation. Accordingly, the ultrastructural examination of the PDGF-treated samples has shown the coexistence of developed RER with ultrastructural traits characteristic of the differentiated state, such as abundant myofilaments connected to well defined dense bodies. On the basis of our observations, we hypothesize that matrix hyperproduction stimulated by PDGF in PSMC may be ascribed to the general enhancement of protein synthesis induced by the agonist, rather than interpreted as resulting from a switch in phenotype. It would be interesting to investigate whether SM--actin and ET-1 receptors together with other markers of the contractile phenotype are up- or down-regulated in peritubular cells of pathological seminiferous tubules affected by or undergoing peritubular hyalinization. Although tubular contractility is an issue of potential clinical relevance, the functionality of myoid cells in such pathological samples appears to be virtually unexplored; to date, there have been only limited data reported indicating loss of desmin (39) and actin (39, 40) immunoreactivity at very late stages of structural alteration. A further point bearing upon the maintenance of SM differentiated state in relation to matrix abundance and composition is that different matrix components have been implicated in either the maintenance or loss of differentiated traits in SMC. Opposing responses have, in fact, been observed in cells plated on surfaces coated with laminin, which has been shown to potentiate the differentiated state (41) through insulin-like growth factor I-induced phosphoinositide 3-kinase activation (34), and fibronectin, which has been found to induce phenotypic modulation to a synthetic state (41). In preliminary experiments, plating of PSMC on dishes coated with either laminin or fibronectin failed to induce evident cellular spreading, orientation, enlargement, or actin reorganization (not shown); the possibility of an indirect action of PDGF on cell shape through the regulated secretion of either of these matrix components therefore seems unlikely.

In response to PDGF, PSMC undergo immediate contraction, followed by progressive hypertrophy
A class of agonists known to enhance -actin synthesis and induce cellular hypertrophy in VSMC is represented by vasoactive peptides, angiotensin, AVP, and ET, three factors that, in long-term treatment, have been found to be responsible for increased size and increased protein synthesis in the absence of proliferation (42, 43, 44, 45). The response of PSMC to prolonged PDGF treatment falls entirely within these specifications. Cytofluorometric analysis of cell size shows an increasing gain in size with progressing treatment, paralleling increased amino acid incorporation. In a previous report (33), PDGF was shown to induce an increase in cell size when administered to postconfluent, nonproliferating VSMC; in that case, though, the increase in size was accompanied by a noticeable decrease in -SM isoactin, whereas the increase observed in myoid cells is more in line with typical vasoconstrictor-induced hypertrophy. Accordingly, PDGF behaves as a contractile agonist for PSMC, capable of inducing both contraction and typical calcium response after acute stimulation (19). The observation that PSMC contraction ensues immediately after PDGF administration argues in favor of a direct response to the agonist, a speculation reinforced by the observation that no known agonist of peritubular contractility has been shown to be produced by this cell type. As for the possible analogies with vascular smooth muscle, in a number of reports PDGF has been indicated as a vasoconstrictor (46, 47), but conclusive evidence for a direct action on VSMC is lacking. In PSMC, overt contraction in response to PDGF-BB is transitory, lasting only a few minutes, after which time the cell shape reverts to a control-like condition, to slowly develop into an elongate and extended conformation. This shape change partially resembles that observed in the same cells after prolonged treatment with ET-1 (10), which suggests common intracellular pathways controlling long-term responses to contraction agonists.

The control of the contractile phenotype and the mechanisms of agonist-stimulated hypertrophy in SMC undoubtedly represent an important field of investigation for the comprehension of relevant vascular pathologies. The heterogeneous composition of the cell population, which may result in uncontrolled selection of cellular subtypes in serum-grown cultures, and the instability of the contractile phenotype in vitro are commonly reported (20, 48) as factors contributing to methodological difficulties in this field of study. As for hypertrophy, additional difficulties are encountered with SMC, as opposed to striated muscle cells, in so far as the former are capable of proliferation, and mitogenic and contractile stimuli may activate interacting pathways in the same cell (48, 49, 50). PSMC do not require the presence of serum in the culture medium and can be cultured in both highly homogeneous populations and in nonproliferating conditions in which the specific response to the addition of single factors can be studied. On these bases we believe that the unexpected hypertrophic, as opposed to hyperplastic, response to PDGF-BB indicates that primary cultures of PSMC are a valid model in which the specific pathway of single agents can be studied in privileged conditions of reduced background interference. In particular, this experimental system may provide the opportunity to investigate intracellular pathways specific to hypertrophy (with respect to hyperplasia) of SMC and thus contribute to the understanding of the mechanisms underlying their pathological behavior.

	Acknowledgments

 
The authors are indebted to Dr. Fabrizio Padula for his valuable support of the flow cytometry experiments, and to Dr. E. Buchdunger (Novartis Pharma) for the generous gift of CGP53716, an inhibitor of PDGF receptor tyrosine kinase activity.

	Footnotes

 
¹ This work was supported by a grant from Ministero per l’Università e la Ricerca Scientifica e Tecnologica (to F.P.).

Received February 4, 2000.

	References

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