Levels Of Keratinocyte Growth Factor Research Articles

Regulation of keratinocyte growth factor and scatter factor in cyclosporin-induced gingival overgrowth.

Epithelial proliferation is a histological characteristic of drug-induced gingival overgrowth. Keratinocyte growth factor (KGF) and scatter factor (SF) are fibroblast-derived growth factors with potent mitogenic and motogenic effects on epithelial cells, and, therefore, could be involved in the pathogenesis of gingival overgrowth. The aims of this study were to investigate: (i) the effects of cyclosporin on KGF and SF expression by gingival fibroblasts; and (ii) the expression levels of KGF and SF mRNA in normal and overgrown gingival tissue. The KGF and SF protein production was determined by enzyme-linked immunosorbent assay. Relative levels of KGF and SF mRNA expression were determined using semi-quantitative reverse transcriptase polymerase chain reaction. Expression levels in biopsies of normal and overgrown gum were also determined. In overgrown fibroblasts, 500 ng/ml cyclosporin significantly inhibited KGF and SF mRNA and protein while 2000 ng/ml cyclosporin induced a stimulatory effect. In normal cells cyclosporin significantly increased both KGF and SF. KGF and SF mRNA was detected in both normal and overgrown tissues with a tendency towards increased expression levels in overgrown tissue. These results suggest that KGF and SF may have an important role in cyclosporin-induced gingival overgrowth.

Keratinocyte growth factor and scatter factor expression by regionally defined oral fibroblasts.

Keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (SF) are two signalling molecules thought to play important roles in regulating epithelial-mesenchymal interactions. Expression of both factors by fibroblasts in subepithelial connective tissue may play a role in maintaining epithelial integrity in health and in the apical migration of junctional epithelium in periodontitis. The aims of this study were (a) to compare expression levels of KGF and SF by periodontal ligament (PDL) and gingival fibroblasts; and (ii) to determine the effects of interleukin (IL)-1 beta, transforming growth factor (TGF)-beta 1, platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF) on KGF/SF expression by these cell populations. Three paired PDL and gingival fibroblast strains were developed. The KGF and SF protein levels were analysed by enzyme-linked immunosorbent assay. Relative levels of KGF and SF mRNA in cytokine-treated cultures were determined using semiquantitative reverse transcriptase polymerase chain reaction. No differences in the levels of KGF and SF produced by PDL and gingival (SOG) populations were found. In both cell types IL-1 beta stimulated KGF and SF expression, while TGF-beta 1 significantly inhibited expression at both the mRNA and protein levels. Epidermal growth factor and PDGF-BB induced differing effects on expression, stimulating SF protein production but inhibiting KGF output in both fibroblast populations. Differences in response to EGF and PDGF were also seen between paired PDL and gingival fibroblasts.

Regulation of thymic epithelium by keratinocyte growth factor

AbstractHere we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that αβ-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3−4−8− thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE–associated chemokine expression in the RAG2−/−thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.

Cell death and proliferation in Nd-YAG laser, electrocautery, and scalpel wounds on mice skin

The purpose of this study is to compare cell death and proliferation in laser, electrocautery and scalpel wounds on the mice epidermis. Wounds were examined by transmission electron microscopy, the detection of free 3′-OH DNA ends and immunohistochemistry of proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), keratinocyte growth factor (KGF) and keratinocyte growth factor receptor (KGFR). Reepithelization was first observed 5 days after scalpel and laser incisions and 7 days after electrocautery incision. Ultrastructurally, keratinocytes in both electrocautery and laser wounds showed similar post-apoptotic necrotic changes. Interestingly, dividing cells were often observed 3 days after laser incision. Apoptotic index in electrocautery wounds was higher than in laser wounds, although there was no significant difference in the PCNA expression level between them. The expression of iNOS, KGF and KGFR in laser wounds was more intense than in electrocautery wounds. In scalpel wounds, keratinocytes did not show significant changes in morphology or of markers of cell death and proliferation during the observation period. Therefore, the increase in the number of dividing cells and in the expression level of iNOS, KGF and KGFR may induce earlier and thicker reepithelization in laser wounds than in electrocautery and scalpel wounds.

Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer

The ability of gonadotropins to act on and regulate normal ovarian surface epithelial (OSE) cells and ovarian cancer cells was investigated. Bovine OSE was used as a model to study normal OSE. Results demonstrate that follicle stimulating hormone (FSH) and the luteinizing hormone (LH) like molecule, human chorionic gonadotropin (hCG), can both stimulate ( 3H)-thymidine incorporation into DNA in normal OSE cells. Similar results were obtained using either purified hormones or recombinant human hormones. A human ovarian cancer cell-line OCC1 was also stimulated to grow in response to FSH and hCG, but the growth of a different human ovarian cancer cell-line SKOV3 was not affected. In addition to effects on cell growth, gonadotropins also stimulated growth factor expression. Both FSH and hCG stimulated steady state levels of keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and kit ligand (KL) mRNA in OSE cells. Previously, KGF, HGF, and KL have been shown to stimulate OSE growth. Both follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were observed in OSE cells by Northern blot analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed on fresh and cultured OSE cells. Normal OSE was found to express FSHR and LHR both in vivo and in vitro. The PCR reaction products were sequenced and found to provide a 100% homology with the bovine gonadotropin receptor sequences previously reported. FSHR and LHR transcripts were also detected in gonadotropin responsive OCC1 cells, but not in the gonadotropin insensitive SKOV3 cells. Observations support the hypothesis that gonadotropins may influence some ovarian cancers. In summary, the current study demonstrates the novel observation that both the FSHR and LHR are expressed by bovine OSE and selected ovarian cancers. Interestingly, the actions of FSH and LH to promote OSE growth may in part be mediated indirectly through an elevation in the expression of autocrine growth factors (KGF, HGF, and KL). Ovarian cancer is more common in conditions with elevated gonadotropins such as post-menopausal women. Therefore, gonadotropin actions on the OSE are postulated to be a potential factor in the onset and progression of some ovarian cancers.

Upregulation of keratinocyte growth factor in the tracheal ligation lamb model of congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) carries a high mortality rate of 60% because of associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. Prenatal tracheal ligation has been shown to improve lung growth in experimental models. This could be caused by a direct effect of increased endothoracic pressure in utero, secondary to the induction of specific growth factors, or both. Keratinocyte growth factor (KGF) is involved in normal lung organogenesis and is a potent mitogen of alveolar type II cells. The authors have therefore investigated the protein and mRNA levels of keratinocyte growth factor in the lung tissue of control, CDH, and CDH tracheal ligation lambs. Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation. Tracheal ligation was performed at 110 days in 4 lambs, and they were delivered by cesarean section at 140 to 145 days. Twin littermates served as controls. The lungs were dissected en bloc and snap frozen. KGF protein levels were determined by ELISA. Total RNA was isolated, and a RNase protection assay was performed using an ovine cDNA probe for KGF, and a human cDNA probe for GAPDH (house keeping control). Densitometric analysis was used to quantify the relative amounts of mRNA in each sample. There was a significant decrease in the KGF protein levels of the CDH samples (110 v 73.2 pg/mg protein, P =.02). This decrease was mirrored by a significant fall in the level of mRNA expression for KGF (0.694 v 0.235, P = .02). Tracheal ligation normalized the KGF protein levels (96.1 pg/mg protein). This elevation of KGF protein was accompanied by an upregulation of KGF gene expression to control levels (0.56). Tracheal ligation clearly is accompanied by an upregulation of keratinocyte growth factor protein and gene expression. It is not yet clear whether keratinocyte growth factor is solely responsible for the growth observed in these tracheal ligation preparations. Further growth factor blocking experiments are required.

KGF AND HGF ARE GROWTH FACTORS FOR MESOTHELIAL CELLS IN PLEURAL LAVAGE FLUID AFTER INTRATRACHEAL ASBESTOS

Mesothelial cells proliferate soon after asbestos deposition in the lung. The present study investigates whether the known mesothelial cell mitogens keratinocyte growth factor (KGF)and hepatocyte growth factor (HGF)are present in the lung and specifically in the pleural cavity during the phase of mesothelial cell growth. Rats received 1 mg crocidolite asbestos in 0.5 mL water by intratracheal instillation and were killed up to 2 weeks later; tritiated thymidine was injected 1 hour before death. Bronchoalveolar lavage (BAL)and pleural lavage (PLL)were performed. Increased inflammatory cell numbers and protein levels were found in BAL but also in PLL at 1 day after asbestos deposition. In lung sections, labeling of mesothelial cells increased > 10-fold at day 1 and stayed above normal for 1 week. During this period, the levels of HGF and KGF were significantly raised in both BAL and PLL fluids. The PLL fluid had mitogenic activity for mesothelial cells in culture and this effect was significantly reduced by antibodies to HGF and KGF. The results indicate that fiber deposition in the airspaces rapidly induces lung injury and inflammation, in which growth factors for mesothelial cells KGF and HGF are secreted. These factors reach the pleural cavity at the time when mesothelial cell proliferation occurs. It is possible that the activated, dividing mesothelial population may then be more susceptible to DNA damage by any translocated fibers.

SERUM KERATINOCYTE GROWTH FACTOR MEASUREMENT IN PATIENTS WITH PROSTATE CANCER

Keratinocyte growth factor (KGF) is a stromally derived growth factor important in mediating androgen induced activities in benign prostatic hyperplasia (BPH) and prostate cancer. We assessed whether serum KGF could be used as a molecular marker in patients with prostate cancer. Using a modified double sandwich enzyme-linked immunosorbent assay, we measured serum KGF in 56 men with prostate cancer and 81 men with BPH. Comparative analyses were made with total serum prostate specific antigen (PSA), disease stage and clinical grade. Following optimization, a sensitive and reproducible assay for serum KGF measurement was developed. Serum KGF levels tend to be higher in men with BPH compared to those with prostate cancer (1,242 and 828 pg./ml., respectively). A weak but significant linear relationship between PSA and KGF (p = 0.034) was found in patients with BPH. There was no association between KGF and tumor grade or stage but there was a strong positive linear relationship between PSA and KGF (p = 0.006, R(2) = 68.3%) in low grade tumors. In those men with serum PSA less than 10 ng./ml. KGF levels were significantly higher in BPH compared to prostate cancer cases (965 +/- 245 and 133 +/- 61.3 pg./ml., respectively, p = 0.0058). Using a KGF threshold range of 500 to 900 pg./ml., specificity for detecting BPH was 88% to 100% and the positive predictive value was 92% to 100%. We have optimized a reproducible and sensitive enzyme-linked immunosorbent assay system for the measurement of serum KGF. Overall KGF levels tend to be lower in patients with prostate cancer than with BPH. In patients with serum PSA less than 10 ng./ml. serum KGF levels were significantly higher in the BPH compared to the prostate cancer group. A large prospective study is indicated to assess the role of serum KGF measurement in patients with prostate cancer.

Expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium and ovarian cancer

The current study investigates the expression and action of keratinocyte growth factor (KGF) in normal ovarian surface epithelium (OSE) and ovarian cancer tissues. Ovarian tumors are primarily derived from the OSE. KGF is a mesenchymal cell-derived growth factor that mediates stromal cell-epithelial cell interactions in a variety of different tissues. Human ovarian tumors from borderline, stage I and stage III cases were found to express KGF protein in the epithelial cell component by immunocytochemical analysis. The stromal cell component of human ovarian tumors contained little or no KGF immunostaining. Normal bovine ovaries have similarities to human ovaries and are used as a model system to investigate normal OSE functions. KGF protein was detected in the OSE from normal human and bovine ovaries by immunocytochemistry. Ovarian stromal tissue contained light but positive KGF immunostaining. RNA was collected from normal bovine OSE and ovarian stromal cells to examine KGF gene expression. KGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. In order to examine and quantitate KGF gene expression in freshly isolated versus cultured tissues, a sensitive quantitative RT-PCR assay for KGF was utilized. KGF gene expression was found to be high in freshly isolated OSE, but very low in freshly isolated stroma. Levels of KGF gene expression after culture of OSE and stromal cells increased. Observations indicate that normal OSE express high levels of KGF in vivo and in vitro. Expression of KGF by normal epithelial cells versus stromal cells was unexpected and suggests KGF may be an important autocrine stimulator of OSE. KGF actions on bovine OSE cells were investigated. KGF was found to stimulate the growth of normal OSE cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to KGF. Current results demonstrate the production and action of KGF on normal OSE cells and ovarian cancer cells. Observations can be interpreted to suggest that KGF may in part be involved in the growth of ovarian tumors. This appears to be one of the first reports of KGF production by an epithelial cell. The autocrine stimulation of OSE growth by the local production and action of KGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

Overexpression of keratinocyte growth factor in cancer cells and enterochromaffin cells in human colorectal cancer.

Keratinocyte growth factor (KGF) is a mitogenic polypeptide that is mainly synthesized by mesenchymal cells. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is localized in epithelial cells. In the present study, the expression level of KGF and KGFR messenger RNA (mRNA), and the localization of these mRNA and proteins in tumor specimens obtained from 12 human colorectal cancer cases were estimated. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the expression of KGF and KGFR mRNA in both colorectal cancer and normal colorectal tissues. In specimens from 10 of the 12 cancer cases, the KGF mRNA level was higher in the specimens obtained from the cancerous portions than in those obtained from non-cancerous tissues of the same cases. KGFR mRNA was higher in cancerous tissues in eight of 12 cases. To localize the KGF protein in normal and cancerous human colorectal tissues, immunohistochemistry was employed. In normal colorectal tissue, faint KGF immunoreactivity was present in a few fibroblasts. In contrast, strong KGF immunoreactivity was present in many of the neuroendocrine cells present in close proximity to cancer cells, and moderate immunoreactivity was recognized in the cancer cells themselves and adjacent fibroblasts. KGF-positive neuroendocrine cells also showed serotonin immunoreactivity, indicating that they were enterochromaffin cells. By in situ hybridization, both KGF and KGFR mRNA were co-overexpressed in these colorectal cancer cells, and KGF mRNA was recognized in neuroendocrine cells lying in close proximity to the cancer cells. These findings indicate the possibility that KGF acts in both a paracrine and autocrine manner to induce colorectal cancer cell growth in vivo.

Effects of keratinocyte growth factor in the endometrium of rhesus macaques during the luteal-follicular transition.

We previously reported that keratinocyte growth factor (KGF) is up-regulated by the action of progesterone (P) in the primate endometrium, and we suggested that this protein is a likely mediator of P-dependent stromal-epithelial paracrine interactions in this tissue. At the end of the menstrual cycle, P levels fall, and the abundance of endometrial KGF transcripts decreases approximately 9-fold. In macaques, withdrawal of P induces the luteal-follicular transition (LFT), marked by menstrual sloughing of the functionalis zone and apoptotic regression of the basalis zone. Because KGF levels fall so dramatically during the LFT, we hypothesized that replacement with exogenous KGF during the LFT would prevent some of the endometrial changes seen after P withdrawal. Here we describe two studies of the effects of exogenously administered KGF during the LFT in rhesus macaques. In one experiment we administered KGF systemically to ovariectomized, juvenile rhesus macaques during an LFT induced by hormonal manipulations. KGF had dramatic proliferative effects on the bladder and salivary glands, known targets of KGF, but did not affect cell proliferation in the endometrium or block menstrual sloughing and bleeding. However, KGF strongly inhibited apoptosis in the basalis zone, increased glandular sacculation and folding in this zone, and had a marked trophic effect on the spiral arteries. In the second experiment we installed oviductal catheters in ovariectomized adult rhesus macaques and infused KGF directly into the uterine lumen during a hormonally induced LFT. Again, arteriotrophic, antiapoptotic, and basalis gland sacculation effects were observed in the absence of any effect on cell proliferation. We concluded that although KGF is mitogenic for many epithelial cell types, it does not play this role in the primate endometrium. Its most important roles may be to stimulate spiral artery growth and inhibit glandular apoptosis during the nonfertile menstrual cycle. Because its expression rises coincident with the time of implantation and because spiral arteries are essential to successful establishment of pregnancy, the role of KGF in the fertile menstrual cycle deserves further study.

Relationship of Keratinocyte Growth Factor and Hepatocyte Growth Factor Levels in Rat Lung Lavage Fluid to Epithelial Cell Regeneration after Bleomycin

Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are known mitogens for normal alveolar Type 2 cells in vitro and in vivo. We wished to determine whether these two growth factors are involved in lung repair after epithelial cell necrosis by determining the levels of each factor in lung lavage fluid collected serially after bleomycin-induced injury, and how these values relate specifically to proliferation of bronchiolar and alveolar epithelial cells. Rats received an intratracheal injection of 1 unit bleomycin in 0.5 ml water and were killed at intervals up to 4 weeks with 1 muCi/g tritiated thymidine injected 1 hour before death. Early necrosis of bronchiolar epithelial (BR) cells and Type 1 alveolar epithelium was followed by an increase in inflammatory cell numbers and high protein levels in bronchoalveolar lavage (BAL) fluids. In addition, the levels of KGF and HGF, measured by enzyme-linked immunosorbent assay in BAL, increased as early as 3 days and peaked at 7-14 days, when KGF was measured at 160 pg/ml (n = 50) and HGF reached 460 pg/ml (n = 40). Both values dropped sharply after 2 weeks. Epithelial cell proliferation was quantitated as percentage of labeled cells in autoradiographs of methacrylate sections. Labeling of BR cells predominated in the first week and peaked at 7% at 3 days. Type 2 cell proliferation was delayed somewhat but occurred in 3 to 10 days with a peak of 7% labeled cells at 1 week. The results demonstrate that both HGF and KGF are present in the lung in greatly increased amounts soon after bleomycin-induced epithelial cell necrosis. These high levels are associated with both BR and alveolar epithelial cell proliferation.

Hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury. Biologic and clinical significance.

Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are among the most potent mitogens identified for alveolar type II epithelial cells and may have other important functions in repair of the alveolar epithelium in acute lung injury (ALI). However, neither growth factor has been identified in the distal air spaces or plasma of patients with ALI. The goals of this study were to determine: (1) whether HGF and KGF are present in pulmonary edema fluid from patients with ALI and control patients with hydrostatic pulmonary edema; (2) whether HGF and KGF are biologically active in pulmonary edema; and (3) whether HGF or KGF levels are associated with clinical outcome. Pulmonary edema and plasma samples were obtained within 48 h of onset of acute pulmonary edema requiring mechanical ventilation in 26 patients with ALI and 11 control patients with hydrostatic edema. HGF and KGF concentrations were measured with enzyme-linked immunosorbent assays (ELISAs). The median (25th to 75th percentiles) concentration of HGF in pulmonary edema fluid was 21.4 (8.3 to 41.3) ng/ml in ALI and 6.6 (4.8 to 11.4) ng/ml in hydrostatic edema fluid (p < 0.01). The HGF concentration was 7-fold higher in the edema fluid than in the plasma of patients with ALI. In contrast, KGF was detected in low concentrations in edema fluid of patients with ALI and hydrostatic pulmonary edema; the concentration of KGF did not differ in ALI edema (0.6 [0.3 to 2.1] ng/ml) and hydrostatic edema fluid (0.2 [0.0 to 2.6] ng/ml) (p = NS). HGF and KGF were partly purified from four edema-fluid samples by heparin-Sepharose chromatography. Partly purified edema fluids were potent stimuli of DNA synthesis in cultured rat type II alveolar cells; addition of neutralizing antibodies to HGF and KGF attenuated this increase in DNA synthesis by 66% and 53%, respectively. Interestingly, higher edema-fluid levels of HGF were associated with higher mortality in patients with ALI. These novel results show that HGF and KGF are active in the alveolar space early in ALI, probably mediating early events in lung repair, and that increased levels of HGF in edema fluid may have prognostic value early in ALI.

Altered expression of epidermal growth factor receptor ligands in tumor promoter–treated mouse epidermis and in primary mouse skin tumors induced by an initiation‐promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor α (TGFα), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFα mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands. Mol. Carcinog. 22:73–83, 1998. © 1998 Wiley-Liss, Inc.

Altered expression of epidermal growth factor receptor ligands in tumor promoter–treated mouse epidermis and in primary mouse skin tumors induced by an initiation-promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Developmental and hormonal regulation of keratinocyte growth factor expression and action in the ovarian follicle.

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5-10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Developmental and Hormonal Regulation of Keratinocyte Growth Factor Expression and Action in the Ovarian Follicle

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5–10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Differential regulation of keratinocyte growth factor and hepatocyte growth factor/scatter factor by different cytokines in human corneal and limbal fibroblasts.

Corneal epithelial stem cells and transient amplifying cells are located in the limbal and corneal regions, respectively. In a serum-free medium with or without different cytokines, limbal fibroblasts consistently produced greater levels of keratinocyte growth factor (KGF) transcript and protein than corneal fibroblasts, whereas corneal fibroblasts produced greater levels of hepatocyte growth factor/ scatter factor (HGF/SF) transcript and protein than limbal fibroblasts. Expression of HGF/SF transcript and protein was up-regulated mildly by epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), or platelet-derived growth factor B (PDGF-BB) but markedly by interleukin-1 beta (IL-1 beta) and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF transcript was down-regulated by EGF, TGF-alpha, and PDGF-BB, was markedly up-regulated by IL-1 beta, and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF protein was up-regulated markedly by IL-1 beta and moderately by PDGF-BB, especially in limbal fibroblasts. TGF-beta 1 uniquely turned off transcript and protein expression of HGF/SF and KGF in corneal fibroblasts. Although its transcript levels were similarly down-regulated in limbal fibroblasts, KGF protein levels were paradoxically up-regulated by TGF-beta 1 when added alone or with TGF-alpha or IL-1 beta. These data indicate that KGF and HGF/SF, two fibroblast-derived epithelial mitogens, are expressed differentially by limbal and corneal fibroblasts and are modulated by cytokines activated during epithelial-mesenchymal interactions, suggesting that they may play a different role in modulating corneal epithelial stem cells and transient amplifying cells.

Keratinocyte Growth Factor and Receptor mRNA Expression in Cholesteatoma of the Middle Ear

Keratinocyte growth factor (KGF) is synthesized and secreted exclusively by stromal cells in epithelialized organs, and specifically promotes proliferation of cells of epithelial origin, including keratinocytes. Cholesteatoma is a proliferative form of keratinocyte dysregulation with aggressive growth that often leads to destruction of adjacent bone. We examined the expression of KGF, closely related peptides and their receptors in the development of cholesteatoma by comparing cholesteatoma with normal ear skin using the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. All surgical specimens of cholesteatoma and most normal ear skin samples expressed detectable levels of KGF and KGF receptor (KGFR). Semiquantitative RT-PCR using β-actin mRNA as an internal standard revealed significantly higher expression of KGF nvRNA in cholesteatoma than in normal skin, while no significant difference in KGFR mRNA expression was noted between cholesteatoma tissue and normal skin. These results suggest that excessive KGF synthesis may contribute to the hyperproliferative state in cholesteatoma.

Detection of keratinocyte growth factor (KGF) messenger ribonucleic acid and immunolocalization of KGF in the canine testis

Keratinocyte growth factor (KGF) was originally discovered in human embryonic lung fibroblasts and is a member of the fibroblast growth factor (FGF) family. Members of the FGF family have been shown to regulate testicular function. However, the recently discovered KGF has not been studied in the testis. KGF has been detected in many other tissues, including the prostate, an organ whose development and function have been associated with presence of the testis. In this study, KGF mRNA was detected in the whole testis using reverse transcription polymerase chain reaction (RT-PCR). The 575-bp KGF-specific product was detected along with a 594-bp β-actin-specific product. To identify the cell types in which KGF mRNA was predominantly expressed, interstitial cells were physically separated from seminiferous tubules. The interstitial cells were then sorted on a discontinuous Percoll gradient and total cellular mRNAs isolated. Using RT-PCR and Southern hybridization with specific cDNA probes, the KGF mRNA was detected in interstitial cells. KGF expression levels were then evaluated semiquantitatively with a competitive RT-PCR assay. KGF expression levels were highest in interstitial cells that equilibrated between 20 and 30% Percoll. Enriched Leydig cells and seminiferous tubules expressed low levels of KGF. Finally, immunohistochemical analysis was performed on canine testes using a rabbit anti-KGF polyclonal antibody. The KGF protein was localized predominantly to peritubular cells of the canine testis. These results suggest that KGF is synthesized in the canine testis.