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Endocrinology Vol. 139, No. 9 3772-3779
Copyright © 1998 by The Endocrine Society

ARTICLES

Keratinocyte Growth Factor Injected into Female Mouse Neonates Stimulates Uterine and Vaginal Epithelial Growth1

Yun Kit Hom, Peter Young, Axel A. Thomson and Gerald R. Cunha

Department of Anatomy, University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Gerald R. Cunha, Department of Anatomy, Mail Stop 0452, University of California, San Francisco, California 94143. E-mail: grcunha{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estradiol (E2) stimulates epithelial growth in the female genital tract via estrogen receptors (ER) in the stroma using paracrine mechanisms. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by mesenchymal cells and is mitogenic for epithelial cells making it a strong candidate as a paracrine mediator. Transcripts for KGF and the KGF receptor were detected in the neonatal mouse uterus and vagina. Treatment of neonatal mice with KGF elicited changes in uterine and vaginal epithelium within five days and induced long term effects in these tissues. Newborn female Balb/c mice were injected daily with 5 µg/g body weight of KGF or saline for five days. KGF-treated mice exhibited a 5- to 6-fold increase in uterine epithelial BrdU-labeling index and a 4- and 5-fold increase in vaginal epithelial BrdU-labeling index vs. respective saline-treated controls. Histological sections of KGF-treated uteri revealed dramatic increases in epithelial surface area due to extensive folding of the luminal epithelium. In some areas, the evaginated luminal epithelium invaded zones normally occupied by myometrium. Vaginal epithelium was thicker than that of saline-treated controls following 5 days of KGF treatment. When KGF-treated newborn mice grew to adulthood and were ovariectomized, vaginal smears exhibited persistent diestrus in all animals. Histologic analysis demonstrated a thick parakeratotic vaginal epithelium (~10 cell layers) 9 days postovariectomy in adult neonatally KGF-treated mice. Our studies indicate that KGF injected into neonates markedly stimulated proliferation of neonatal uterine and vaginal epithelium and elicited long-term, persistent abnormal changes in vaginal epithelium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS maintain function, stimulate growth, and are obligatory for normal epithelial cytodifferentiation and secretory activity in the female genital tract. Specifically, estrogen induces DNA synthesis in uterine and vaginal epithelium (1) by acting through estrogen receptors (ER) to stimulate estrogen specific transcription of genes required for physiologic responses. Because ER in the adult uterus are expressed in the epithelium, stroma, and myometrial cells (2, 3), estradiol may elicit epithelial growth directly via epithelial ER or indirectly via stromal ER through paracrine mechanisms.

It was initially assumed that the effects of E2 on epithelium and stroma were mediated directly through ER in these tissue compartments. However, analysis of ER expression and E2 responsiveness in the neonatal mouse uterus raised doubt concerning this interpretation. Immunocytochemical and steroid autoradiographic studies demonstrated that in neonatal mice, ER was undetectable in uterine epithelium (UtE) but were present in uterine mesenchyme (UtM) (4, 5) during fetal and neonatal periods when the uterus is undergoing organogenesis. Despite the apparent lack of uterine epithelial ER in neonatal mice, injection of diethylstilbestrol (DES) increased the rate of UtE proliferation (6). From these studies, it was proposed that the mitogenic effects of estrogens on neonatal UtE were elicited via paracrine growth-promoting influence from ER-expressing mesenchymal cells. This interpretation has been recently verified by tissue recombinant studies employing wild-type and estrogen receptor-{alpha} knockout (ERKO) mice (7).

Certain growth factors such as KGF are known to be paracrine mediators of stromal-epithelial interactions. KGF has been shown to be expressed in the mesenchyme of several developing organs, whereas the KGF receptor is expressed only in epithelia (8, 9). In various developing hormone target organs KGF has been shown to be a paracrine mediator of mesenchymal/epithelial interactions. Exogenous KGF was able to replace androgens in eliciting growth of the seminal vesicle and prostate (10, 11). Furthermore, epithelial growth and development were inhibited by neutralizing KGF with a monoclonal antibody or a soluble KGF receptor peptide. KGF has been shown to be induced by androgens in prostatic stromal cells in vitro (12) but does not appear to be regulated by androgens in vivo (13). Because KGF appeared to act as an epithelial morphogen and mitogen in the seminal vesicle and prostate, it is possible that KGF could mediate epithelial growth in a similar manner in estrogen target organs of the rodent female reproductive tract. In this regard, KGF has been previously shown to stimulate growth of rodent mammary epithelium both in vivo (14) and in vitro (15). Furthermore, KGF is induced in monkey endometrium in response to progesterone suggesting that KGF is a "progestomedin" (16).

Our objective in this study was to examine both short- and long-term effects of exogenous KGF on the developing female mouse urogenital tract. Our results indicate that KGF can acutely stimulate epithelial proliferation in the neonatal mouse uterus and vagina. In addition, neonatal treatment with KGF elicits ovary-independent persistent vaginal hyperplasia in adulthood. The effects produced by KGF treatment were very similar to those elicited by estradiol, suggesting that KGF may be an important mediator of paracrine signaling in the female reproductive tract.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Normal female Balb/c mice 0–2 days old were obtained from the Cancer Research Laboratory, University of California (Berkeley, CA). All animals were maintained in accordance with NIH Guide for Care and Use of Laboratory Animals, and all procedures described here were approved by the UCSF animal care and usage committees. Mice were maintained under controlled temperature and lighting conditions during the experiment, and were given food and water ad libitum. This study is based on the analysis of 44 KGF-treated and 6 saline-treated control mice.

Injections
For short-term experiments, newborn female Balb/c mice (<=2 days old) were injected sc on their dorsal side with recombinant KGF (gift from Amgen) (5 µg/g body weight per day) or saline as a control for 5 days. Body weights for each neonate were recorded before each injection. One day after the last injection, mice were killed and their uteri and vaginae removed. For cell labeling studies, mice were injected ip with 0.1 mg/g BW bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) 2 h before they were killed.

For long-term experiments, newborn female Balb/c mice were injected sc on their dosal side with KGF (5 µg/g body weight per day) or saline as a control for 3 days. At 60 days, the neonatally KGF-treated mice and saline controls were ovariectomized and maintained for another 9 days during which vaginal smears were taken daily.

Microdissection
For short-term experiments, KGF-treated and saline-treated control neonates were killed 1 day after the final injection, and the entire genital tract was excised. Images of the freshly dissected uteri and vaginae were captured using a digital Lumina camera (Leaf System, South Brough, MA) to document gross morphology.

Vaginal smears
Just before ovariectomy at 60 days and each day for 9 days thereafter, the vaginae of adult mice were flushed with a small volume (~50 µl) of sterile PBS using a Pasteur pipette. Vaginal aspirates were spread across microscope slides, air dried and stained with hematoxylin.

Histology
Female neonatal mouse genital tracts described above were fixed, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin. In a similar manner, uteri and vaginae from adult mice were also fixed, embedded in paraffin, sectioned at 6 µm, and stained.

Immunocytochemistry for BrdU assay
Sections on slides were deparaffinized, rehydrated to 70% ethanol, and rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 10 min followed by DNA denaturation in 2 N HCl for 30 min both at 37 C. After rinsing thoroughly in PBS, slides were treated with 0.4% pepsin in 0.01 N HCl for 5 min at 37 C. Slides were blocked with 5% sheep serum for 15 min at 37 C after which the sections were incubated for 1 h at 37 C with the avidin-biotin conjugated anti-BrdU antibody (Zymed, South San Francisco, CA) at 1:40 dilution. After rinsing the slides with PBS, sections were processed with Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). The avidin-biotin complex was then developed with 0.05% diaminobenzidine for approximately 4 min and rinsed thoroughly with tap water. After counterstaining with hemotoxylin, slides were coverslipped and analyzed for BrdU labeling indices.

Labeling index
Epithelial labeling index with BrdU was determined as the percentage of labeled epithelial cells in the total population of epithelial cells as described previously (17). Individual histological sections to be scored were selected randomly, and for a given section only regions of appropriate section orientation were scored in which the plane of section was roughly perpendicular to the plane of the basement membrane. Areas of poor section quality, tangential or oblique orientation were excluded. For both uteri or vaginae, a minimum of 300 cells were scored per individual specimen in 3–6 replicate specimens per treatment group.

RNA analysis
RNA was prepared from neonatal vaginae and uteri by homogenization in RNA Stat-60 (Tel-Test "B" Inc., Friendswood TX). RNAse protection assay was performed as previously published by our group (13). Briefly, 10 µg of total RNA was used in all experiments. Total RNA was incubated with [32P]UTP-labeled antisense riboprobes overnight at 45 C in hybridization buffer (80% formamide, 0.4 M NaCl, 40 mM PIPES, pH 6.6, 1 mM EDTA, pH 8.0). After hybridization, 350 µl of digestion buffer (0.5 M NaCl, 10 mM Tris, pH 7.5, 5 mM EDTA, pH 8.0, 5 U RNAse A, 200 U RNAse T1, Ambion, Austin, TX) was added and samples were incubated at 30–37 C for 45 min. After the addition of 30 µg Proteinase K and 20 µl 10% SDS, the samples were further incubated for 15 min at 37 C. Samples were extracted with phenol/chloroform and then chloroform followed by ethanol precipitation. Samples were loaded onto a 6% acrylamide 8 M urea sequencing gel after denaturation at 94 C for 4 min in 80% formamide gel loading buffer. The gel was dried and exposed to DuPont reflection x-ray film with an intensifying screen at -80 C for 16 h. KGF transcripts produced a band of 227 nucleotides and KGFR transcripts produced a band of 150 nucleotides in the RNAse protection assay.

Statistics
Values are expressed as the mean ± SEM of at least six different experiments. Differences among means were estimated using a Student’s unpaired t test and ANOVA. Differences were considered significant at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of KGF and KGFR in the newborn mouse uterus and vagina
RNA was isolated from pooled uteri and vaginae of neonatal mice. By RNase protection assay, transcripts for KGF and the KGFR were detected in total RNA in whole uteri and vaginae of 4- to 5-day-old mice (Fig. 1Go).



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  		Figure 1. Analysis of transcripts for KGF and KGFR in whole vaginae and uteri from normal, untreated 4- to 5-day-old Balb/c mice. Total RNA was prepared from homogenized whole vaginae and uteri, and 10 µg were used in an RNAse protection experiment with 32P-labeled antisense riboprobes for KGF, KGFR, and cyclophilin (as a control for RNA integrity and loading differences). KGF and KGFR mRNA were detected in both vaginal and uterine organs.

 
Wet weight gain of KGF- and saline-treated female mouse neonates
Mouse neonates were weighed just before initiation of treatment and daily thereafter. Weight gain of the KGF-treated mice fell behind those of saline-treated control neonates. By the end of the 5-day injection period, KGF-treated mice weighed approximately 30% less than saline-treated mice. The weight deficiency of KGF-treated mice occurred in spite of the fact that milk was found in their digestive tracts.

Gross morphology of KGF- and saline-treated female neonatal genital tracts
Figure 2Go depicts micrographs of the entire female neonatal mouse genital tract freshly dissected from a control saline-injected neonate and KGF-treated neonate (five daily injections, 5 µg/g body weight/day). Although overall size of genital tracts from both experimental conditions was similar, the KGF-treated uteri showed an elaborately folded epithelium compared with the control (Fig. 2CGo). Gross morphological differences were not observed between KGF- and saline-treated vaginae.



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  		Figure 2. Micrograph of entire female neonatal mouse genital tract freshly dissected from A) control saline injected neonate, and B) KGF-treated neonate. Although overall size of genital tracts from both treatment groups are similar, the KGF-treated genital tract shows an elaborate, folded uterine epithelial morphology compared with the control. Panel C is a higher magnification of the uterus from the KGF-treated neonate.

 
Histology of KGF- and saline-treated neonatal uteri and vaginae
Histological sections of uteri and vaginae of control and KGF-treated neonatal mice injected on days 1–5 postnatal are presented in Fig. 3Go. Histology verified the extensive epithelial folding in KGF-treated uteri suggested by the wholemount photos (Fig. 3BGo). In some areas, the evaginated uterine epithelium had invaded zones normally occupied by myometrium. This degree of epithelial growth in the KGF-treated neonatal uterus was particularly striking and was highly unusual. KGF also stimulated thickening of the vaginal epithelium which was also parakeratotic (Fig. 3DGo).



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  		Figure 3. Carnoy’s fixed, paraffin embedded, and hemotoxylin/eosin stained sections of A) control neonatal uterus, B) KGF-treated neonatal uterus, C) control neonatal vagina, and D) KGF-treated neonatal vagina. The extensive epithelial evagination suggested by the wholemount micrographs of the KGF-treated uterine epithelia in Fig. 2Go are confirmed histologically. Normal uterine myometrium is identified by arrows in Panel A. As indicated by arrows in Panel B, the evaginated luminal epithelium has invaded zones normally occupied by myometrium. KGF also stimulated parakeratotic differentiation in the neonatal vaginal epithelium shown in Panel D.

 
Neonatal uterine and vaginal epithelial labeling indices
Uterine and vaginal epithelial labeling indices were determined as the percentage of BrdU-labeled epithelial cells per total basal epithelial cells (Fig. 4Go). KGF elicited a 5- to 6-fold increase in uterine luminal epithelial cell labeling index and a 4- to 5-fold increase in vaginal basal epithelial cell labeling index in comparison to saline-treated controls. Differences were significant at P <= 0.05. No noticeble growth effect was observed in either the uterine or vaginal stroma in response to KGF treatment presumably because KGF receptors were not present.



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  		Figure 4. BrdU labeling indices for epithelium of control and KGF-treated uteri and vaginae. Epithelial labeling index percentages were determined by counting the number of BrdU labeled epithelial cells divided by total number of epithelial cells normalized to 100. Each bar is the mean of at least six measurements with SEM. *, P < 0.05.

 
Long-term effects of neonatal KGF treatment
KGF-treated female neonates were also allowed to live to adulthood. All neonates injected with KGF (5 µg/g body weight) on days 1 to 5 died in the week following the last injection. For that reason the dosage of KGF was reduced to three injections (5 µg/g body weight on days 1 to 3). Survival was 67% out of an initial group of 15 neonates treated at this dosage. Upon reaching adulthood (60 days), the neonatally KGF-treated and saline-treated control mice were ovariectomized. At the time of ovariectomy, vaginal smears of all adult neonatally KGF-treated mice exhibited an abundance of leukocytes, mucinous strands and nucleated epithelial cells indicative of diestrus (Fig. 5Go). Smears were not taken from KGF-treated mice before ovariectomy. However, it is unlikely that diestrus-smears obtained from all KGF-treated mice at ovariectomy were due to estrus cycle synchrony because these animals were housed in different cages. Furthermore, vaginal smears from saline-treated controls represented all stages of the estrus cycle (not illustrated). Such diestrus vaginal smears produced by KGF-treated mice were maintained continuously for 9 days post ovariectomy suggesting persistent alterations of the vaginal epithelium. Following ovariectomy of saline-treated mice, vaginal smears contained few cells (not illustrated). At 9 days after ovariectomy, mice were killed, and their vaginae and uteri were examined histologically. Vaginal sections of neonatally KGF-treated mice ovariectomized in adulthood exhibited a thick parakeratotic epithelium (~10 cell layers thick) 9 days postovariectomy (Fig. 6Go). In contrast, neonatally saline-treated mice ovariectomized in adulthood exhibited an atrophic epithelium that was 2–3 layers in thickness. Uteri of adult neonatally KGF-treated mice were histologically normal in all respects.



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  		Figure 5. Photograph of vaginal smear from ovariectomized adults of neonatally KGF-treated mice. The KGF dosage was three injections (5 µg/g body weight) on days 1–3. KGF-treated and saline-treated newborns were aged to adulthood (~60 days) and then were ovariectomized. At the time of ovariectomy and afterwards, vaginal smears of all adult neonatally KGF-treated mice exhibited an abundance of leukocytes, mucinous strands, and nucleated epithelial cells indicative of persistent diestrus. The vast majority of cells are leukocytes with a few nucleated and anucleated epithelial cells (representatives as indicated).

 


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  		Figure 6. Histological sections of (A) vaginae and (B) uteri from KGF-treated mice ovariectomized at 60 days. Neonates injected with KGF (5 µg/g body weight) on days 1–3 were aged to adulthood (60 days) and then ovariectomized. Nine days after ovariectomy, mice were killed and vaginae and uteri were dissected. Organs were then fixed, paraffin embedded, sectioned at 6 µm, and stained with hematoxylin and eosin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While growth factors have been named for their effects on cellular proliferation, further experimentation has revealed a range of effects on other phenomena. This is certainly true for KGF, which in addition to being an epithelial mitogen also has effects on epithelial morphogenesis and differentiation as discussed below. In hormone target organs of male and female reproductive tracts, KGF has been proposed to be an epithelial mitogen of mesenchymal/stromal origin. In the male genital tract, androgens are known to elicit epithelial proliferation via paracrine signals from the mesenchyme/stroma (18). In this regard, KGF has been shown to be mitogenic for prostatic and seminal vesicle epithelium and to be able to elicit orderly ductal growth in the neonatal rat prostate (10, 11, 12, 19). Likewise in the mammary gland, tissue recombinant studies with ER-negative (ERKO) and wild-type mice have recently shown that mammary ductal growth is elicited via paracrine signals from mammary stroma (20). KGF and the KGFR are expressed in the mammary gland, and KGF has been shown to stimulate mammary epithelial proliferation both in vitro (15) and in vivo (21). Therefore, based upon previous reports and the present study, we propose that KGF is a paracrine mediator in estrogen/progestin target organs such as mammary gland, uterus and vagina, particularly since KGF can elicit effects in these organs that mimic actions of estradiol. Recent studies have shown that estrogen elicits proliferation of uterine and vaginal epithelium through ER in stromal cells via paracrine mechanisms (7, 22). The present study demonstrates the presence of transcripts for KGF and KGF receptor in the neonatal mouse uterus. The mitogenic effect of KGF on uterine and vaginal epithelia supports the concept that KGF may be an important paracrine mediator of estrogen action in the female genital tract. Finally, based upon expression patterns and up-regulation of KGF by progesterone, KGF has been suggested to be a "progestamedin" in the monkey uterus (16). Future studies will determine if KGF transcripts and protein are directly or indirectly up-regulated by estradiol. Direct regulation of KGF by estradiol would imply that the KGF gene promoter is regulated by the estrogen receptor even though estrogen response elements have not been identified in the KGF promoter. Equally possible is the scenario in which estradiol indirectly up-regulates KGF. For example, estradiol may induce a growth regulator whose action is to indirectly elicit synthesis and secretion of KGF by stromal cells. An indirect mechanism of regulating KGF expression in the female genital tract is favored because KGF is known to be broadly expressed in the mesenchyme of many hormone target and nonhormone target organs. Clearly, in most organs KGF regulation occurs via mechanisms unrelated to estrogen or progesterone receptors.

The mitogenic effect of KGF on uterine or vaginal epithelium is presumably elicited through an isoform of the fibroblast growth factor receptor 2, a tyrosine kinase receptor, which binds KGF, acidic FGF and possibly a few other members of the FGF ligand family (23, 24, 25, 26). The KGF receptor has been shown to be restricted to epithelia (8, 9). The importance of signaling through this receptor has been demonstrated in experiments in which epithelial growth is markedly impaired by a dominant negative form of this receptor (27, 28). In the present study, transcripts for KGF receptor were detected in the mouse uterus and vagina by RNase protection. Thus, the in vivo effect of KGF on uterine and vaginal epithelium is presumed to be elicited via KGF receptor in these epithelia.

In addition to mitogenic effects, KGF also mediates morphogenetic effects on epithelium. KGF and KGFR mRNA were detected in lung mesenchyme and epithelium, respectively, of 13 day fetal rats in which bronchial branching morphogenesis is particularly active (8, 9, 29, 30, 31). Bronchial branching morphogenesis is severely disrupted in transgenic mice engineered to overexpress a dominant negative form of the KGF receptor in the lung (27). Furthermore, another transgenic mouse overexpressing KGF displayed dilated cystic bronchi (32). Thus, it appears that proper morphogenesis requires the presence of precise levels of KGF. In this study, death of neonates injected with KGF for 5 days may have been due, at least in part, to impaired pulmonary function. In vitro studies have shown that embryonic lung rudiments cultured with antisense oligonucleotides to either KGF or KGFR exhibited impaired lung bronchial branching morphogenesis; this effect was reversed by exogenous KGF (30). However, in another study using a similar culture system, KGF by itself was found to reduce the number of terminal branches and to elicit formation of cyst-like bronchial structures (31). While KGF has been shown by in situ hybridization to be uniformly expressed in the mesenchyme of developing lungs, another FGF (FGF10) has recently been shown to be expressed in mesenchyme surrounding growing distal lung buds (33).

Organ cultures have been used to examine the role of KGF in ductal branching morphogenesis in the rodent seminal vesicle (10) and prostate (11). Neutralization of KGF with monoclonal antibodies or a soluble KGF receptor peptide inhibited testosterone-induced ductal branching morphogenesis in organ cultures of both the mouse seminal vesicle (10) and rat prostate (11). This inhibition was attributed at least in part to reduced epithelial proliferation. Significantly, the requirement for androgens in ductal branching morphogenesis could be satisfied by KGF itself. A similar morphogenic effect of KGF has been reported for the mammary gland. Newborn rats injected with KGF displayed abnormal dilated cystic mammary ducts (21). Transgenic mice with overexpression of KGF targeted to the mammary gland displayed mammary epithelial hyperplasia and inappropriate alveolar morphogenesis (34). Likewise in the present study, the pattern of uterine epithelial morphology was affected by KGF, which elicited extensive folding of the uterine luminal epithelium of the newborn female mouse (see Figs. 2Go and 3Go). Evagination of the uterine epithelium of the KGF-treated mice was so pronounced that the epithelium encroached into the area normally occupied by myometrium. This abnormal epithelial growth seen in KGF-treated neonates was apparently reversible since uteri of adult neonatally KGF-treated mice did not exhibit adenomyosis.

KGF is also known to regulate epithelial differentiation. In the stomach of fetal rats, and mice KGF and KGFR transcripts have been detected in the mesenchyme and epithelium, respectively (9, 35) indicating that KGF might mediate epithelial-mesenchymal interactions in gastric epithelial development even though the expression of KGF and KGFR was not concurrent with epithelial proliferation during the morphogenic period. Instead, KGF expression occurred after the morphogenic period of gastric development indicating that KGF may also function as a differentiation factor. The retarded weight gain observed in KGF-treated neonates may have been due to abnormal differentiation of gastrointestinal epithelium. For epithelial tissues such as those in the lung and intestine, several distinct subsets of highly differentiated epithelial cells arise during development. For instance, the intestine contains enterocytes, goblet cells, endocrine cells, and paneth cells. Each of these cell types is localized to a particular morphologic niche, and in normal tissues each cell type constitutes a certain percentage of the total number of epithelial cells for that tissue. Studies in which KGF was injected into adult rats and have shown a disproportionate increase in the relative number of mucinous goblet cells in the intestinal epithelium (36). Whether this effect is due to an enhanced mitogenesis of goblet cells themselves or whether this effect is mediated through a direct influence of KGF on the differentiation pathway of a progenitor cell is unknown. In either case, the net result for the intestinal epithelium is a marked enhancement of the proportion of goblet cells, presumably at the expense of other epithelial cell types. Similar phenomena may be applicable to the lung in which KGF can increase the proportion of type 2 alveolar cells. In the case of the lung, enhanced numbers of type 2 alveolar cells appear to result from specific mitogenesis of these cells (37).

KGF clearly affects keratinocyte terminal differentiation and/or programmed cell death in culture (38, 39). Overexpression of KGF in transgenic mice induced global abnormalities in skin differentiation (40). KGF expression targeted to the epidermis under the control of the human keratin 14 promoter produced transgenic mice with altered epidermal growth and differentiation including wrinkled skin, increased epidermal thickness, and suppression of hair follicle morphogenesis. In rat prostatic organ cultures, KGF stimulated the production of prostate-specific secretory proteins (DP-1 and probasin) which are markers of prostatic epithelial differentiation (41). In the case of the vagina, KGF appears to promote epithelial cornification, an effect normally elicited by estrogen (42). Vaginal cornification results from two separate processes: proliferation to generate a thick epithelial layer and cornification which involves the expression of keratins 1 and 10 and other differentiation markers (43, 44, 45, 46, 47). Vaginal epithelial proliferation is elicited by mitogens of stromal origin (7) KGF being a likely candidate. The parakeratosis observed in the present study in mice treated with KGF indicates that like the epidermis, KGF may directly regulate vaginal epithelial keratinization. Supporting this conclusion is the finding that KGF elicits squamous metaplasia in a human prostatic epithelial cell line in vitro (48).

While our study suggests a function for KGF in the female genital tract, KGF null female mice are fertile and morphologically normal except for defects in skin and hair follicles (49). Because defects in the female genital tract were not observed in KGF knockout mice, KGF clearly is not the sole mediator of estrogen-dependent stromal-epithelial interactions. It is possible that other members of the FGF family (perhaps FGF10) or growth factors were able to compensate for the loss of KGF.

Female rodents treated with estradiol or diethylstilbestrol (DES) during the early neonatal period become anovulatory and subsequently develop persistent vaginal hyperplasia and cornification in adulthood (50). This constellation of endocrine abnormalities is due in part to permanent perturbation of the hypothalamic-pituitary-gonadal axis as well as in part on direct effects of estrogen on the developing vagina (51). The direct effect of exogenous estrogen on the vagina is thought to be mediated by estrogen receptors, which are detectable in vaginal stroma at birth. Epithelial ER are expressed shortly after birth in the vagina (4). Given the recent tissue recombination studies using wild-type and ERKO tissues, it is now evident that certain estrogenic effects on vaginal, uterine, and mammary epithelia are mediated through paracrine mechanisms (4, 20, 22, 52). Thus, it is appropriate to speculate whether the induction of ovary-independent persistent vaginal hyperplasia is due to direct effects of estrogen mediated via epithelial ER, or whether the induction of ovary-independent persistent vaginal hyperplasia is a paracrine event elicited via stromal ER through paracrine mediators. Based upon tissue recombinant studies, paracrine mechanisms have been proposed (53). Our current findings support the concept that paracrine mediators may in fact be involved in the induction of ovary-independent persistent vaginal hyperplasia in so far as KGF given neonatally resulted in persistent vaginal epithelial changes in adulthood. Effects elicited by KGF included persistent diestrus and ovary-independent persistent vaginal epithelial hyperplasia in neonatally KGF-treated adult mice. These effects in neonatally KGF-treated mice were remarkably similar to those elicited by estradiol or DES in newborn mice (54). Exogenous estrogens may induce persistent vaginal hyperplasia via paracrine mediators such as KGF that permanently disrupt epithelial differentiation during periods of organogenesis. Ovary-independent persistent vaginal hyperplasia elicited neonatally by exogenous estrogen treatment is a highly abnormal condition that can progress to more severe epithelial atypias such as dysplasia and carcinoma (51). The results of this study suggest that KGF may be involved in initiating this cascade of pathological events.


   Footnotes
 
1 This research was supported by Grant AG-13784. Back

Received January 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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