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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 6 2186-2191
Copyright © 1998 by The Endocrine Society

Original Studies

Expression and Cellular Localization of Keratinocyte Growth Factor and Its Receptor in Human Hyperplastic Prostate Tissue1

Alessandra De Bellis, Clara Crescioli, Cecilia Grappone, Stefano Milani, Paola Ghiandi, Gianni Forti and Mario Serio

Department of Clinical Physiopathology, Endocrine Unit (A.D.B., C.C., P.G., G.F., M.S.) and Gastroenterology Unit (C.G., S.M.), University of Florence, Florence, Italy

Address all correspondence and requests for reprints to: Dr. Alessandra De Bellis, Department of Clinical Physiopathology, Endocrine Unit, Viale Pieraccini 6, 50139 Florence, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well recognized that the actions of androgens alone do not explain the hyperplastic development of the gland that occurs in elderly men. The increasing number of reports confirming the lack of mitogenic activity of androgens coupled with the powerful mitogenic activity of growth factors and the discovery of growth factor receptors led to an increased interest in the putative role of growth factors in prostate physiopathology. We have previously demonstrated the presence and the cellular localization of epidermal growth factor and of the related peptide, transforming growth factor-{alpha}, together with their common receptor in the epithelial compartment of the human hyperplastic prostate tissue (BPH). In the present study we examined the expression and cellular localization of messenger ribonucleic acid (RNA) encoding keratinocyte growth factor (KGF) and its receptor in human hyperplastic prostate tissue. RT-PCR of total RNA extracted from BPH tissues documented the presence of transcripts for KGF and its receptor. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that KGF mRNA was mainly localized in the stromal cells, whereas its receptor was mainly localized in the prostate epithelium. Moreover, the mitogenic activity of KGF on cultured BPH cells compared to that of other growth factors has been tested. Our findings clearly indicate that KGF has the ability to function as a potent mitogen in BPH cells. Our data support the hypothesis that KGF plays an important role in prostate growth and that in human prostate it seems to act in a paracrine fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DEVELOPMENT of the prostate and maintenance of adult structure and function are controlled by androgens. However, a large body of evidence indicates that androgen may not act directly, but may act indirectly through polypeptide growth factors (1).

We have previously demonstrated the presence of specific receptors for epidermal growth factor (EGF) (2) and insulin like growth factor I (IGF-I) in human hyperplastic prostate tissue [benign prostate hyperplasia (BPH)] (3) the localization of IGF-I receptors in the basal layer of the prostate epithelium (3) and their up-regulation by androgen deprivation (2, 3). Furthermore, we have demonstrated the IGF-I expression and its predominant stromal localization in BPH tissue (4). More recently, we have shown the expression of messenger ribonucleic acid (mRNA) for EGF, transforming growth factor-{alpha}, and their common receptor, EGF receptor, in BPH tissue and their localization in the epithelial cells (5).

Keratinocyte growth factor (KGF), a member of the fibroblast growth (FGF) family, is expressed in stromal fibroblasts and acts specifically on cells of epithelial origin as a paracrine mediator (6). FGFs regulate a wide variety of biological activities, including embryonic development, wound repair, and angiogenesis, and have been implicated in various diseases, including malignant transformation (7). KGF was initially purified and cloned from a lung fibroblast line as a soluble factor that could stimulate keratinocyte proliferation (8).

The KGF receptor (KGF-R), a membrane-spanning tyrosine kinase, is an alternatively spliced isoform of FGF-2 (bek/FGFR2) (9, 10) that binds acidic FGF with equally high affinity and basic FGF (bFGF) with much lower affinity (11, 12). Expression of KGF transcript has been detected in several stromal fibroblast cell lines derived from epithelial tissues of embryonic, neonatal, and adult human sources (6). In vivo recombinant KGF (rKGF) was found to induce the proliferation of hair follicles, sebaceous glands, and regenerating keratinocytes within rabbit dermal wounds (13). Moreover, rKGF was shown to induce the proliferation of type II pneumocytes in adult rats (14), of hepatocytes (15), and of epithelial cells from the foregut to the colon (16) and urothelium (17) and to stimulate pancreatic ductal epithelia as well as mammary gland ductal epithelia (18, 19). Moreover, KGF appears to be an important mediator of the epithelial-mesenchimal interactions required for androgen-dependent seminal vesicle development in mice (20).

Taken together, these observations imply that KGF is an endogenous paracrine effector for a variety of epithelial cells that is synthesized by underlying stromal fibroblasts. The present results indicate that KGF is probably an important mediator of cell growth and differentiation in the human prostate, and that the administration of KGF has a highly significant inductive effect on specific epithelial cells within this organ.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

Prostatic tissues for RNA extraction and in situ hybridization were obtained from patients who underwent suprapubic adenomectomy for BPH. No pharmacological treatment was performed in the 3 months preceding surgery.

After surgery, the tissues were put in liquid nitrogen and stored at -80 C until processing. Normal term (36–42 weeks gestation) placentas were obtained immediately after spontaneous vaginal delivery or uncomplicated cesarean section. The study was approved by the local institutional review.

Cell culture

BPH cells were established from freshly delivered prostate tissue obtained from patients with BPH at surgery. Tissue was cut into small fragments and treated overnight with 2 mg/mL bacterial collagenase (~700 U/mL). Fragments were than extensively washed in phosphate-buffered saline (PBS) and cultured in growth medium (MEM supplemented with 10% heat-inactivated FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin). Cells began to emerge within 1 week and were used within the fifth passage. Cells in BPH cell cultures were a mixture of fibroblasts (for the most part) and fibromuscular cells. Media, antibiotics, and collagenase type IV were supplied by Sigma Chemical Co. (St. Louis, MO), and FBS was obtained by Unipath (Milan, Italy). Plasticware was purchased by Falcon (Oxnard, CA), and disposable filtration units were purchased from PBI International (Milan, Italy). Human rKGF, bFGF, and EGF were purchased by Boehringer Mannheim (Mannheim, Germany).

RNA isolation and RT-PCR

Total RNA was extracted from 10 BPH tissues by single step guanidine thiocyanate-phenol-chloroform extraction (21). RNA samples were quantified by their absorbance at 260 nm and by ethidium bromide staining of samples electrophoresed on agarose gel. Total RNA was reverse transcribed into complementary DNA (cDNA) by RT-PCR, following a procedure previously described (5). The sequences of the upstream and downstream oligonucleotide primers for KGF and KGF-R amplification are reported in Table 1Go. The amplification reactions consisted in denaturation at 95 C for 90 s, annealing at 60 C, and extension at 72 C for 90 s for 30 cycles for KGF and KGF-R. The reverse transcribed products were amplified by PCR using 12.5 pmol each of a specific primer pair for ß-actin, as previously described (5). PCR analyses were repeated three or four times for each sample.


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  		Table 1. Sequence of the oligonucleotide primers used for KGF and KGF-R amplification

 
In situ hybridization

For in situ hybridization, cryostat sections (4–7 µm thick) were dried on a hot plate at 80 C, fixed in 4% paraformaldehyde-PBS, pH 7.4, for 20 min, and washed three times in PBS (22). Complementary RNA probes were obtained by run-off transcription of the 324-bp EcoRI/BamHI fragment of the human KGF cDNA clone, the 148-bp EcoRI/HindIII PCR fragment of the human KGF-R DNA subcloned into the appropriate restriction sites of the plasmid pT3/T718U (Pharmacia, Piscataway, NJ; both provided by Drs. Rubin and Finch) (22). After linearization of the plasmids with the appropriate restriction endonuclease, T7 or T3 RNA polymerase (Boehringer Mannheim) were employed to obtain either the antisense or sense (negative control) strands, respectively. Transcription, labeling of RNA probes prehybridization, hybridization, removal of nonspecifically bound probe by ribonuclease A digestion, and further washing procedures were performed for positive and negative strand RNA probes as discussed in detail previously (23).

Cell proliferation assay

For growth measurement, 55 x 103 cells (15 x 103 cell/cm2) were seeded onto 12-well plates in growth medium. After 24 h, the growth medium was removed, and the cells were accurately washed in PBS and incubated in phenol red-free medium containing 0.5% BSA and human rKGF at the following concentrations: 0.1, 1, 10, 25, 50, and 100 ng/mL. Cells in phenol red-free medium containing 0.5% BSA were used as control. After 48 h, cells were trypsinized and counted by a hemocytometer. All experiments were performed in double and repeated at least three times. The results are expressed as cell number per mL.

Statistical analysis

Statistical analysis was performed using Student’s test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and cellular localization of KGF and KGF-R

To study KGF and KGF-R expression in BPH tissue, we used the sensitive technique, RT-PCR. Figure 1Go represents an ethidium bromide-stained agarose gel, showing the products of RT-PCR using the specific oligonucleotide primers for KGF. A transcript with the predicted size of 266 bp was amplified in all tissues examined. All samples, including the positive control, represented by human placenta (10) gave an amplification product at apparently similar abundance. As a control for RNA integrity and RT-PCR procedure, the same RNA samples were also analyzed for ß-actin expression, and all samples showed an amplification product of the expected size of 741 bp at apparently similar abundance.



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  		Figure 1. Analysis of KGF expression in BPH and placental tissues by RT-PCR. Top panel, Ethidium bromide-stained agarose gel, showing the presence of the 266-bp RT-PCR product using specific primers for KGF. Lanes 1–5 represent different BPH specimens; lane 6 represents one placenta specimen; lane 8 is the negative control in which reverse transcribed cDNA was omitted from the reaction mix. Molecular mass markers are indicated in lane 7. The size of the predicted amplified product is also indicated. Bottom panel, Ethidium bromide-stained agarose gel showing the presence of the expected RT-PCR product for ß-actin in all tissue samples tested for KGF expression.

 
RT-PCR products for KGF-R are shown in Fig. 2Go. The amplified product of the predicted size of 187 bp was detected in all tissue samples at apparently similar abundance, including the positive control represented by human placenta (10). The expected RT-PCR product for ß-actin mRNA was present in all samples examined with apparently comparable abundance (not shown).



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  		Figure 2. Analysis of KGF-R expression in BPH and placental tissues by RT-PCR. Ethidium bromide-stained agarose gel, showing the presence of the 187-bp RT-PCR product using specific primers for KGF-R. Lanes 1, 2, 3, 5, and 6 represent different BPH specimens; lane 7 represents one placenta specimen; lane 8 is the negative control in which reverse transcribed cDNA was omitted from the reaction mix. Molecular mass markers are indicated in lane 4. The size of the predicted amplified product is also indicated.

 
To identify the cellular sources of KGF and KGF-R mRNAs, we examined four different BPH specimens by in situ hybridization with specific RNA probes. For KGF mRNA, the hybridization signal was very abundant and mainly localized in the stromal compartment (Fig. 3Go, A and B) of all tissues examined. The hybridization signal for KGF-R was also abundant and localized in the epithelial compartment (Fig. 3Go, C and D). The specificity of in situ hybridization was confirmed by hybridizing tissue sections with the respective labeled sense KGF and KGF-R probes (Fig. 3Go, E and F).



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  		Figure 3. In situ hybridization of BPH tissue with KGF and KGF-R mRNA probes. A and C, Representative BPH tissue sections hybridized with the labeled antisense mRNA probes for KGF and KGF-R, respectively (magnification, x400); B and D, higher magnification (x1000). E and F, Control BPH tissue section hybridized with 35S-labeled sense KGF and KGF-R mRNA probes, respectively (magnification, x400).

 
Growth responsiveness of BPH cells to hKGF, bFGF, and EGF

The addition of human rKGF to BPH cell cultures resulted in a significant increase in the proliferation rate, starting from 1 ng/mL as shown in Fig. 4Go. At 10 ng/mL rKGF, the maximum effect in terms of cell proliferation was obtained. Similarly, when basic FGF was added to BPH cell cultures, a significant increase in cell proliferation was noted at 1 ng/mL, and at a concentration of 10 ng/mL, the effect on cell growth was increased. The mitogenic potency of EGF was also tested in cultured BPH cells. EGF at 10 ng/mL was not able to significantly stimulate cell growth (Fig. 5Go).



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  		Figure 4. Effect of rKGF on BPH cell proliferation. KGF was added to cultured BPH cells at the indicated concentrations for 48 h. The mean ± SD are indicated. The experiment was performed at least three times, and the figure is representative. *, P < 0,01.

 


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  		Figure 5. Effects of bFGF and EGF on BPH cell proliferation. FGF and EGF were added to cultured BPH cells at the indicated concentrations for 48 h. The experiment was performed at least three times, and the figure is representative. *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates for the first time the expression and cellular localization of mRNAs encoding KGF and KGF-R in human hyperplastic prostate tissue. Specific transcripts for KGF and KGF-R were detected in all tissues examined using the highly sensitive technique RT-PCR. In situ hybridization studies demonstrated that in BPH tissues, KGF mRNA was abundant and appeared to be predominantly expressed in stromal cells, whereas KGF-R mRNA was also well represented and mainly localized in prostatic epithelium.

Members of the FGF family are direct acting growth regulators of isolated prostate cells (24) and, therefore, candidates for mediators of the indirect control of epithelial cells by androgen. Expression of members of the FGF receptor family in the human prostate has been also investigated, and there is evidence that their expression is similar in normal and hyperplastic prostate (24). Although there are not many data regarding the expression of KGF and its receptor in human normal, hyperplastic, and neoplastic prostate, it is known that KGF expression, its binding characteristics, and mitogenic activity on cell cultures are not very different whether cultures are initiated from normal or hyperplastic prostates (25), and human fetal prostate tissues do not express mRNA transcripts for KGF and KGF-R (26). FGF family members have biological properties that could contribute to the transformed phenotype, but their exact role in prostate cancer remains unclear. In a recent study by Leung et al. (27), it has been demonstrated that KGF mRNA was expressed in prostate cancer, but its expression appears to be unrelated to either tumor grade or progression. It is of interest that prostate epithelial cells express the splice variant of the FGF receptor 2 (bek) receptor gene that exhibits specificity for KGF (10), and this variant appears to be undetectable in the epithelium from androgen-independent malignant prostate tumor. Although the significance of this observation remains to be clarified, this change in the expression of FGF receptor isoforms potentially underlies the stromal independence and progressive autonomy of malignant androgen-independent tumor cells. Therefore, additional studies are required to clarify the expression of FGFs and their receptors in the different prostate cell types in both normal and pathological conditions.

Studies of Cunha and colleagues have established the important role of KGF in epithelial induction during mouse seminal vesicle development (20), an androgen-dependent process. In fact, besides being a potent mitogen for epithelium, KGF has been shown to dramatically influence branching morphogenesis in the seminal vesicle. The addition of a KGF-specific neutralizing antibody caused a striking inhibition of seminal vesicle growth and branching morphogenesis, and when KGF was substituted for testosterone in culture medium, seminal vesicle growth was about 50% of that observed with an optimal dose of androgen. Interestingly, the same studies carried out using neonatal ventral prostate cultures (28) have demonstrated that the addition of KGF to the androgen-deficient medium of prostate cultures was able to elicit growth and ductal morphogenesis comparable to those elicited by testosterone. Thus, for prostatic development, the requirement for testosterone could be totally replaced by KGF.

The KGF signaling pathways appear to contribute not only to the normal regulation of prostate growth and differentiation, but also to the regulation of other tissues, such as the human endometrium and myometrium (29), and the corneal epithelial cells (30). Interestingly, KGF mRNA expression has also been detected in breast tumors (31). Although a role for KGF in mammary gland development remains speculative, the presence of KGF mRNA in breast tumors and the demonstration of its mitogenic activity on mammary epithelium (31) suggest that this growth factor could contribute to epithelial cell proliferation in the breast.

Our data demonstrate the greater stimulatory potency of KGF on cultured BPH cell proliferation compared to that of other growth factors, such as bFGF and EGF. In fact, the addition of rKGF to BPH cell cultures determined a significant increase in cell proliferation starting from 1 ng/mL, with a maximum effect at 10 ng/mL, whereas bFGF at the same concentration used for KGF appeared to be less potent in BPH cell proliferation. In contrast, EGF at 10 ng/mL was not effective on BPH cell growth. Notably, Culig et al. investigated the effect of some growth factors on stimulation of androgen receptor (AR) gene transcription in human prostate cell lines (32). IGF-I, KGF, and EGF directly activate AR in the absence of androgen, and IGF-I was even more potent than KGF, which, in turn, was more effective than EGF. It is possible that at least part of these growth stimulatory effects is due to activation of the AR, suggesting the existence of a signaling pathway between growth factors and AR that needs to be further investigated. We have also tested the stimulatory potency of des(1, 2, 3)-IGF-I on BPH cell growth. Although it is known that des(1, 2, 3)-IGF-I is a more potent stimulator of cell growth than IGF-I because it binds to IGF binding proteins with lower affinity (34), our data provide evidence that this factor stimulates the proliferation of BPH cells starting from a concentration of 0.1 ng/mL, thus more efficiently than all other growth factors tested (not shown). Notably, previous studies have shown that IGF-I displayed the strongest stimulation on the AR-mediated reporter gene transcription regardless of the nature of the androgen-inducible promoter (33). Although the mechanism of prostate growth is still poorly understood, mesenchymal-epithelial interactions are known to play a critical role in the development of androgen target organs. Growth factors and their receptors appear to play an important role in this process, with both paracrine and autocrine pathways involved. KGF as well as IGF-I (4) might exert their effects with a paracrine mode of action, with both produced by stromal cells and their receptors localized in the prostatic epithelium. In particular, our data support the hypothesis that KGF is a direct acting growth regulator of cultured BPH cells and, therefore, a candidate mediator of the indirect control of epithelial cell proliferation by androgen.


   Acknowledgments
 
The authors are grateful to Drs. J. S. Rubin and P. Finch, who kindly provided the plasmids containing KGF and KGF-R cDNAs.


   Footnotes
 
1 This work was supported by the National Research Council (Program Senescence 94.00433.40), Applicazioni Cliniche Ricerca Oncologica, and Associazione Italiana Ricerca sul Cancro. Back

Received July 2, 1997.

Revised March 11, 1998.

Accepted March 18, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chevalier S, Beauregard I, Defoy LT, et al. 1996 Action, localization and structure-function relationship of growth factors and their receptors in the prostate. Reprod Med Rev. 5:73–205.
  2. Fiorelli G, De Bellis A, Longo A, et al. 1989 Epidermal growth factor receptors in human hyperplastic prostate tissue and their modulation by chronic treatment with a gonadotropin-releasing hormone analog. J Clin Endocrinol Metab. 68:240–243.
  3. Fiorelli G, De Bellis A, Longo A, et al. 1991 Insulin like growth factor-I receptors in human hyperplastic prostate tissue: characterization, tissue localization, and their modulation by chronic treatment with a gonadotropin-releasing hormone analog. J Clin Endocrinol Metab. 72:740–746.[Abstract]
  4. Barni T, Vannelli BG, Sadri R, et al. 1994 Insulin like growth factor-I (IGF-I) and its binding protein IGFBP-4 in human prostatic hyperplastic tissue: gene expression and its cellular localization. J Clin Endocrinol Metab. 78:778–783.[Abstract]
  5. De Bellis A, Ghiandi P, Comerci A, et al. 1996 Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization. J Clin Endocrinol Metab. 81:4148–4154.[Abstract/Free Full Text]
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