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Keywords:

  • Calponin;
  • myosin heavy chain;
  • sm22;
  • smoothelin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

ABSTRACT: Smooth muscle (SM) is an important component of the prostatic stroma. We previously showed that, despite extensive morphologic changes, smooth muscle cells (SMCs) of the rat ventral prostate preserve some differentiation markers 21 days after castration. In the present study, we investigated whether the expression of SMC markers is preserved in the rat ventral prostate after long-term castration. Adult Wistar rats were castrated and sacrificed 100 days after surgery. The ventral prostates were processed for histology, stereology, immunocytochemistry (SM α-actin and SM-myosin heavy chain [MHC]), transmission electron microscopy (TEM), and reverse transcription polymerase chain reaction (smoothelin, sm22, and calponin). The prostates of castrated rats showed significant weight reduction, corresponding to only 5.6% of the control. Stereology showed that SMCs occupied the same proportion of the prostate volume but suffered a significant reduction in absolute volume (5.5% of control). The SMCs were retracted and showed spinous outlines. TEM revealed the presence of an abundant myofibrillar component, dense plaques, and an external lamina in these cells. SMCs were reactive to antibodies against SM α-actin and SM-MHC and expressed mRNA for smoothelin, sm22, and calponin. The results confirmed that rat prostatic SMCs are affected by androgen deprivation. Although showing marked phenotypic changes, these cells expressed SMC markers at the protein (SM α-actin and SM-MHC) and mRNA (smoothelin, sm22, and calponin) levels. These observations support the idea that SMCs may modulate their phenotypes (contractile vs synthetic) without changing their differentiation states.

The prostate gland largely depends on androgens for its embryonic and pubertal development and for the maintenance of adult activity (Isaacs et al, 1994). Surgical or chemical castration causes a marked involution of the organ, which is mainly attributed to a decrease in epithelial cell activity and apoptosis. The stroma is also subjected to marked changes after androgen deprivation, occupying a growing fraction of the organ volume. In the stroma, in addition to a reduction in blood vessels, increase in vascular permeability (Shabsigh et al, 1998, 1999), and extracellular matrix reorganization, mainly related to elastic fibers (Carvalho et al, 1997), basement membrane (Carvalho and Line, 1996), and collagen fibers (Vilamaior et al, 2000), the functions of cellular components are also reprogrammed. Among these components, smooth muscle cells (SMCs) are prominently affected. Previous studies have suggested that prostatic SMCs progressively dedifferentiate after castration. This suggestion arose from the observation that the myofibrillar fraction is reduced in favor of an increase in the volume fraction occupied by organelles of the secretory pathway, indicating that these cells undergo a contractile to synthetic phenotypic change (Zhao et al, 1992; Niu et al, 2001, 2003). It was also shown that SMCs sequentially lose some molecular markers, mirroring their appearance during morphogenesis, suggesting that the phenotypic modulation is coupled to dedifferentiation (Hayward et al, 1996).

More recently new markers have been proposed to be specific for SMCs (Doevendans and van Eys, 2002). Using some of these markers, we showed that, in addition to extensive morphologic changes including an increase in the volume density of organelles of the synthetic secretory pathway (Vilamaior et al, 2005), SMCs exhibit many of the morphologic landmarks of differentiated cells and preserve SM myosin heavy chain (MHC) and smoothelin expression up to 21 days after castration (Antonioli et al, 2004). This finding led us to propose that the contractile to synthetic phenotypic change does not involve dedifferentiation.

We further argued that the discrepancy between our results and those reported by Hayward et al (1996) might be attributable to the length of the androgen deprivation period (21 vs 100 days, respectively) and/or the use of distinct differentiation markers (myosin, vinculin, desmin, and laminin vs smoothelin and MHC) and then decided to investigate whether long-term castration results in the loss of these newer and more specific markers of SMC differentiation. Pursuing this task, we investigated the expression of SM α-actin and SM-MHC (at the protein level) and smoothelin, sm22, and calponin (at the mRNA level), as well as morphologic, ultrastructural, and stereologic alterations, to determine the effects of long-term androgen deprivation on the differentiation state of SMCs in the rat ventral prostate. The results allowed us to confirm that, despite showing morphologic changes, SMCs preserve their differentiated state after long-term castration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Animals and Histologic Processing

Fifteen 3-month-old male Wistar rats were used. Ten animals were subjected to orchiectomy by means of a scrotal incision under chloral hydrate anesthesia. Ventral prostates were removed 100 days after surgery. Five age-matched rats were used as controls. The protocol was approved by the Committee of Ethics on Animal Experimentation from the State University of Campinas (protocol 1223–1).

The ventral prostates were dissected out, weighed, and immediately fixed by immersion in 4% formaldehyde in phosphate-buffered saline for 24 hours. The samples were then washed, dehydrated, cleared in xylene, and embedded in Paraplast Plus embedding medium (Oxford Labware, St Louis, Mo) for immunocytochemistry. Some fragments were partially dehydrated and embedded in Historesin (Leica Microsystems, Heidelberg, Germany) for general morphology. Stereology was performed as described previously (Antonioli et al, 2004; Garcia-Florez et al, 2005). Volume density (Vv) was calculated as the percent of points in Weibel grid falling on SMC. The total SMC volume was calculated as a product of volume density per prostatic weight, considering the specific gravity of the prostatic tissue as 1.0 (Huttunen et al, 1981). The results were compared using the Student's 2-sample t-test.

Transmission Electron Microscopy

Tissue fragments (∼1 mm3) were processed for transmission electron microscopy. In brief, the fragments were fixed for 24 hours in a solution containing 0.25% tannic acid and 3% glutaraldehyde in Millonig buffer, postfixed in 1% osmium tetroxide for 1 hour, and then incubated in 0.5% uranyl acetate in maleate buffer overnight before dehydration in a graded acetone series and embedding in Araldite (Polysciences Inc, Warrington, Pa). Ultrathin sections were contrasted with lead citrate. Analysis and documentation were carried out with a Leo 906 transmission electron microscope (Zeiss, Oberkochen, Germany).

Immunocytochemistry

Five-micrometer sections were mounted on silanized glass slides, dewaxed with xylene, and rehydrated in a decreasing ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in water for 30 minutes. Nonspecific protein-protein interactions were blocked by incubation with 3% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, Mo) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 hour. Monoclonal antibodies against SM α-actin (A2547; Sigma-Aldrich) and SM-MHC (sc-6956; Santa Cruz Biotechnology, Santa Cruz, Calif) were diluted 1:50 and 1:100, respectively, in TBS-T containing 1% BSA and applied to sections for 1 hour at room temperature. After three 5-minute washes with TBS-T, the sections were incubated with a peroxidase-conjugated antibody against mouse polyvalent immunoglobulins (Sigma-Aldrich) diluted 1:100 in 1% BSA in TBS-T for 1 hour. Sections were washed again, and peroxidase activity was developed with 3,3′-diaminobenzidine followed by counterstaining with methyl green, air drying, and mounting in Entellan (Merck, Darmstadt, Germany). For SM-MHC immunocytochemistry, sections were pretreated with 0.4% pepsin in 0.01 N HCl for 30 minutes at 37°C before endogenous peroxidase blocking. The specimens were observed under a Zeiss Axioskop microscope and photographed using Kodak 100 Proimage film (Rochester, NY).

Reverse Transcription Polymerase Chain Reaction

The prostates were dissected out, weighed, and immediately homogenized with a Polytron in TRIzol (Invitrogen, Carlsbad, Calif). Total RNA was then extracted according to the instructions provided by the manufacturer. The amount of RNA was determined by measuring the absorbances at 260 and 280 nm using a correspondence factor of 40. For cDNA synthesis, reverse transcription (RT) was performed using SuperScript III reverse transcriptase (Invitrogen) for 60 minutes at 50°C and for 15 minutes at 70°C. Polymerase chain reaction (PCR) was carried out with 150 ng of cDNA in a final volume of 25 μL containing Taq polymerase PCR master mix (Promega Corporation, Madison, Wis) and 0.6 pmol of the following primer sets (Invitrogen) under the following reaction conditions: β-actin forward 5′-CTGGCCTCACTGTCCACCTT-3′, reverse 5′-AGTACGATGAGTCCGGCCC-3′, annealing temperature 64°C, 30 cycles; smoothelin forward 5′-GTCGACATCCAGAACTTCCTCC-3′, reverse 5′-CGCAGGTGGTTGTACAGCGA-3′, annealing temperature 94°C, 35 cycles (Rensen et al, 2002); calponin forward 5′-GAAGATCAATGAGTCAACCG-3′, reverse 5′-TGTTCTCAAACAGGTCGTTGGC-3′, annealing temperature 61.5°C, 30 cycles; and sm22 forward 5′-AGGTCTGGCTGAAGAATGGC-3′, reverse 5′-TTCAAAGAGGTCAACAGTCTGG-3′, annealing temperature 60°C, 30 cycles. The sizes of the reaction products for β-actin, smoothelin, calponin, and sm22 were 64, 440 (visceral) and 330 (vascular), 150, and 200 bp, respectively. PCR products were analyzed by 2% agarose gel electrophoresis in Tris-acetate-EDTA buffer and visualized by ethidium bromide staining. The 50-bp DNA step ladder (Promega Corporation) was used as a marker.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Table 1 shows the body and prostate weights and the effect of androgen deprivation for 100 days on these parameters. There was no variation in body weight, whereas prostate weight showed a 20-fold reduction. Accordingly, the relative weight of the ventral prostate was reduced to just 5.5% of the control.

Table 1. . Body and prostatic weight variation 100 days after castration (means ± SD)
 Control (n = 5)Castrated (n = 10)
  1. *Statistically significant at P < .001

  2. †Calculated by dividing prostatic weight by total body weight

Body weight, g449.0 ± 36.3420.5 ± 35.9
Ventral prostate weight, g*0.421 ± 0.0460.021 ± 0.006
Relative weight*†0.089 ± 0.0160.005 ± 0.002
Percent variation−94.4

Androgen deprivation resulted in marked remodeling of the rat ventral prostate. In addition to marked epithelial modifications, hematoxylin and eosin staining revealed that control SMCs were elongated and flattened against the epithelial basement membrane, showing slightly irregular outlines. Following castration, shortening of the cell was observed accompanied by a pleating of the cell surface, leading to a spinous aspect (Figure 1A and B). The SMCs apparently lost the contact between each other and presented with wider extracellular spaces. Usually more than 1 layer of SMCs was found below the epithelial structures after castration.

image

Figure 1. . Hematoxylin and eosin (H & E) staining of a Historesin section and immunocytochemistry for smooth muscle α-actin and myosin heavy chain. H & E staining of the ventral prostates of control animals (a) showed elongated smooth muscle cells (smc) usually present as a single layer below the epithelium (Ep), with apparent contacts between each other. In contrast, in castrated rats, the smooth muscle cells were markedly retracted and atrophic, showing a spinous aspect with very thin processes and forming multiple layers below the epithelium, and were mainly separated from each other by the presence of extracellular matrix (b). Smooth muscle α-actin (c—e) and myosin heavy chain (f—h) were detected in smooth muscle cells of control (c and f) and castrated rats (d, e and g, h). Whereas the staining for actin was more diffuse throughout the cells, that for myosin heavy chain was more concentrated in some regions of the cell, especially around the nucleus. Bar = 10 μm.

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Stereologic analysis showed that androgen deprivation for 100 days did not result in any modification of the prostatic volumetric fraction (Vv) occupied by SMCs (Table 2). However, considering the large reduction in prostatic weight, long-term castration caused a significant decrease in absolute SMC volume (ie, SMC volume corresponded to only 5.5% of that calculated for the age-matched control) (Table 2).

Table 2. . Stereologic analysis of smooth muscle cells in the rat ventral prostate 100 days after castration (means ± SD)
 ControlCastrated
  1. *P < .001

Volume density, %9.92 ± 2.7011.05 ± 3.67
Absolute volume, mL × 10−24.18 ± 1.130.23 ± 0.08*

Transmission electron microscopy revealed important aspects of the SMCs, which were elongated and exhibited slightly irregular outlines (Figure 2A through C). In cross sections, the irregular outline of the cell nucleus, scarcity of organelles, dense plaques, and presence of an external (basal) lamina were noted (Figure 2B and D). The SMCs were separated from the epithelium by a single fibroblast layer (Figure 2C). Androgen deprivation promoted marked SMC reorganization, the most prominent change being retraction of the cytoplasm, which resulted in marked irregular outlines. Myofilaments were the predominant element of the cytoplasm (Figure 2E). Collagen fibrils were closely encrusted in the grooves of the SMC surface (Figure 2E through G). Fibroblasts were also found to extend thin processes among the collagen fibrils in the SMC infoldings (Figure 2F). The external lamina, subplasmalemmal dense plaques, and abundant caveoli, 3 major structural components of SMCs, were preserved after castration (Figure 2G). Although most SMCs were separated from one another, close contacts were maintained at certain points (Figure 2G).

image

Figure 2. . Transmission electron microscopy of smooth muscle cells (SMCs) in the ventral prostates of control and castrated rats. SMCs are flat and elongated, showing slightly undulated outlines in noncastrated rats (a). In cross sections, further morphologic aspects, such as the irregular outline of the cell nucleus, predominance of myofibrils in the cytoplasm, and presence of dense plaques and an external lamina, are readily observed (b). In control animals, the cells were elongated and separated from the base of the epithelium by a single fibroblast layer (c). The detail in (d) shows the presence of an external lamina (asterisks) surrounding the SMCs, subplasmalemmal dense plaques (arrows), and abundant caveoli. In castrated rats, the SMCs are retracted and exhibit highly folded surfaces, contributing to the spinous aspect, and a cell nucleus preserving the irregularity of the nuclear surface. Collagen fibrils are intimately associated with the grooves of the cell surface (e). The detail in (f) shows that, in addition to collagen, fibroblast processes dig deep into the grooves on the SMC surface. Although usually separated, the SMCs of castrated rats (g) show some points of cell-cell adhesion (arrows). EC indicates endothelial cell; Ep, epithelium; F, fibroblasts and/or fibroblast processes; smc, smooth muscle cell; col, collagen fibrils; and M, mitochondria. Bars: a = 10 μm; b = 2 μm; c, e, and g = 5 μm; d = 0.5 μm; f = 1 μm.

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In addition to the morphologic and ultrastructural characterization of the effects of long-term androgen deprivation, we also investigated the expression of some SMC markers. Immunocytochemistry revealed that SM α-actin (Figure 1C through E) and SM-MHC (Figure 1F through H) were present in the SMCs of control rats (Figure 1C and F, respectively) and that androgen deprivation for 100 days had no effect on this expression pattern (Figure 1D and E, and 1G and H, respectively). Moreover, RT-PCR showed that smoothelin, calponin, and sm22 were expressed at the mRNA level in the ventral prostate of control and castrated rats (Figure 3). The expression of the visceral isoform of smoothelin (Figure 3B) was identified.

image

Figure 3. . Reverse transcription polymerase chain reaction using mRNA extracted from the prostates of control and castrated rats. The integrity of the cDNA preparation was confirmed with a primer set for β-actin. A 64-bp amplicon was identified in control and castrated rats and in the urinary bladder. The reaction for smoothelin amplified 2 bands, a vascular (430 bp) and visceral (330 bp) isoform from both control and castrated animals. The same was observed for calponin and sm22, which were detected in control and castrated rats, and presented amplicons of 150 bp and 200 bp, respectively. Smoothelin mRNA was identified even 100 days after castration. Ct indicates control; Cs, 100 days after castration; and Bld, bladder.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

SMCs exert a common contractile function throughout the body. These cells differ in terms of contractility, part of them showing a phasic contraction pattern and the remaining ones are tonic. Furthermore, in contrast to cardiac and skeletal muscle, these cells do not terminally differentiate (ie, they might be recruited for further proliferation and moreover for performing different functions). These complex patterns of functioning might result from the intricate embryonic origins of SMCs (Gittenberger-de-Groot et al, 1999).

Vascular SMCs have received more attention than the prostatic ones. However, prostatic SMCs are also important because of their central role in benign prostatic hyperplasia (Shapiro et al, 1992) and because they seem to be important in regulating epithelial function and behavior, including in cancer (Cunha et al, 1996; Hayward and Cunha, 2000). The primary response of prostatic SMCs to castration is related to the fact that they express the androgen receptor (Prins et al, 1991; Hayward and Cunha, 2000).

We previously examined the behavior of prostatic SMCs in castrated rats and observed that these cells undergo extensive morphologic changes (Antonioli et al, 2004) and present an increase in secretory organelles (Vilamaior et al, 2005) after androgen deprivation for 21 days. We also showed that these cells express SM α-actin, MHC, and smoothelin (Antonioli et al, 2004), suggesting that these cells undergo marked phenotypic modifications in association with distinct functions (Vilamaior et al, 2000) but preserve their differentiation states. This assumption is in perfect agreement with the idea of Owens et al (2004) on vascular SMC phenotype modulation (or switching). On the other hand, this proposal contradicts a previous report by Hayward et al (1996), who studied the differentiation of SM during prostatic development as well as changes related to androgen deprivation and concluded that the modifications reported for the latter reproduced the developmental acquisition of differentiation markers. This discrepancy might be attributed to the use of different differentiation markers, as well as to the investigation of different periods of androgen deprivation.

The present study was then idealized to reproduce the long-term castration experiment of Hayward et al (1996) using the same markers as employed before (Antonioli et al, 2004), in addition to a detailed morphologic study of SMCs. We showed that SMCs occupy a volume twice that of the prostate in these aged animals as compared with 90-day-old animals (Antonioli et al, 2004) and that they became markedly atrophic after 100 days of androgen deprivation. Although SMCs occupied the same volume fraction of the organ (∼10%) with respect to the age-matched controls, they showed a marked reduction in the total volume, considering the extreme reduction in prostatic weight. At the ultrastructural level, SMCs were readily recognized by the presence of abundant myofilaments, external lamina (basal lamina) and subplasmalemmal dense plaques. These cells were also reactive to anti—SM α-actin and anti—SM-MHC, in addition to expressing smoothelin, sm22, and calponin. While the last 2 markers might reflect the presence of vascular SM, the expression of the visceral (or urogenital) isoform of smoothelin (Rensen et al, 2002) is probably restricted to prostatic SMCs.

It is possible that a fraction of the SMCs do indeed dedifferentiate and contribute to the reduction in the total SMC volume in the prostate of castrated animals, in addition to the death of some and the atrophy of the remaining differentiated cells. This question will require precise methods for labeling and counting SMCs for different periods after castration.

It is not well known to what extent prostatic SMCs are comparable to vascular SMCs. However, it has been shown for the latter that the quiescent (and contractile) state is due to the expression and activation of cAMP response element—binding protein (CREB) and that the transition to a proliferative and migratory phenotype involves CREB inactivation (Reusch and Watson, 2004). Other transcription factors probably involved in this transition are GATA6 and GAX (favoring the quiescent state) and basic transcription element—binding protein 2 and Egr-1 (favoring the proliferative state), whereas serum response factor and myocyte enhancer factor 2 would have dual effects on SMC behavior (Walsh and Takahashi, 2001). Myocardin is another important factor regulating the expression of SMC-specific molecules (Owens et al, 2004). Furthermore, the specific arrangement of CArG elements (CC[AT]6GG motif) and their variations within the promoter region of genes encoding sm22, MHC, and α-actin are likely to be responsible for setting the cell type specificity and temporal expression of SMC-specific genes (Owens et al, 2004). The expression and function of these transcription factors have not been investigated in prostatic SMCs and thus deserve future study.

Another important question in the biology of prostatic SMCs is whether the alterations in SMC phenotype observed after androgen deprivation are comparable to those seen in cancer invasion. Researchers have studied the modifications in SMC behavior during stromal activation in response to cancer progression and emphasized that SMCs undergo progressive dedifferentiation to myofibroblasts (Cunha et al, 1996; Tuxhorn et al, 2001, 2002; Wong and Tam, 2002). Whereas the use of cell cultures is of limited applicability since the mere placement of SMCs in culture is sufficient to promote phenotypic changes, the lack of appropriate experimental models limits the progress in this area.

In conclusion, we demonstrated that prostatic SMCs undergo phenotypic modulation (or switching) upon androgen deprivation while preserving major differentiation markers. It should be emphasized that the phenotypic modulation of SMCs upon androgen deprivation cannot be seen as a passive response but should be considered to be an active adaptation to the new hormonal condition as well as to the changing stromal (and organ) microenvironment, with possible contributions of altered levels of transforming growth factor β and basic fibroblast growth factor (Niu et al, 2003).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
Footnotes
  1. Supported by FAPESP (grants 99/11365–1 and 03/08653–8), CAPES and CNPq.