Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Fibroblast growth factor-10. A second candidate stromal to epithelial cell andromedin in prostate

Fibroblast growth factor (FGF)-10, a homologue of FGF-7, is expressed significantly in normal rat prostate tissue, well differentiated rat prostate tumors with an epithelial and stromal compartment and only in derived prostate stromal cells in culture. Similar to FGF-7, recombinant rat FGF-10 was a specific mitogen for prostate epithelial cells. In contrast to FGF-7 which is widely expressed among stromal cells in tissues, the expression of FGF-10 correlated with the presence of stromal cells of muscle origin. Radioreceptor binding assays and covalent cross-linking analysis revealed that FGF-10 binds with an affinity equal to FGF-7 to resident epithelial cell receptor, FGFR2IIIb, but unlike FGF-7 also binds the IIIb splice variant of FGFR1. Analysis of mRNA expression by RNase protection revealed that, similar to FGF-7, the expression of FGF-10 was responsive to androgen in stromal cells from normal prostate and non-malignant differentiated tumors. Although FGF-10 cDNA exhibits a signal sequence for secretion, cultured stromal cells exhibit strictly a cell-associated FGF-10 antigen that correlates with an alternately translated intracellular isoform. FGF-10 requires 1.4 times higher NaCl for elution from immobilized heparin than does FGF-7 and binds to four times the number of sites on the pericellular matrix of epithelial cells. The results show that prostate stromal cell-derived FGF-10, like FGF-7, exhibits the properties of an andromedin which may indirectly mediate control of epithelial cell growth and function by androgen. Although FGF-10 and FGF-7 bind and activate the same resident epithelial cell receptor (FGFR2IIIb), differences in cell type of origin, compartmentation by alternate translation, the affinity for FGFR1IIIb, and access to FGFR by differential interaction with pericellular matrix heparan sulfate suggest they may play both independent and compensatory roles in prostate homeostasis.     

Keratinocyte growth factor: expression by endometrial epithelia of the porcine uterus

Keratinocyte growth factor/fibroblast growth factor-7 (KGF/FGF-7) is an established paracrine mediator of hormone-regulated epithelial growth and differentiation. In all organs studied, KGF is uniquely expressed in cells of mesenchymal origin. To determine whether KGF and its receptor, keratinocyte growth factor receptor (KGFR) or fibroblast growth factor receptor-2IIIb, were expressed in the porcine uterus as a potential paracrine system mediating progesterone action, we cloned KGF and KGFR partial cDNAs from the porcine endometrium. KGF and KGFR expression was detected in endometrium by Northern blot hybridization. Interestingly, in situ hybridization results demonstrated that KGF was expressed by endometrial epithelia and was particularly abundant between Days 12 and 15 of the estrous cycle and pregnancy. KGF secretion into the lumen of the porcine uterus was also detected on Day 12 of the estrous cycle and pregnancy. KGFR was expressed in both endometrial epithelia and conceptus trophectoderm. These novel findings suggest that KGF may act on the uterine endometrial epithelium in an autocrine manner and on the conceptus trophectoderm in a paracrine manner in the pig, which is the only species possessing a true epitheliochorial type of placentation.     

Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice.

During mammalian embryogenesis, epithelial-mesenchymal interactions play a determining role in normal tissue patterning and development. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a mesenchymally-derived mitogen for epithelial cells. As the KGF receptor is expressed by epithelial cells of numerous tissues and KGF is produced in adjacent stromal cells, KGF is thought to play a role in mediating epithelial cell behaviour. To further investigate the role of this molecule in the development of ocular epithelia we employed transgenic mice engineered to overexpress human KGF in the eye. The most striking phenotypic development was the hyperproliferation of embryonic corneal epithelial cells and their subsequent differentiation into functional lacrimal gland-like tissues. This indicates that stimulation of the KGF receptor early in development, in surface ectoderm normally destined to form corneal epithelium, is sufficient to alter the fate of these cells. Furthermore, this suggests that the correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal gland induction. These transgenic mice provide a valuable model system to study the mechanisms underlying cell fate decisions during ocular morphogenesis.     

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P- labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P- induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P- dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.     

p21-activated protein kinase 4 (PAK4) interacts with the keratinocyte growth factor receptor and participates in keratinocyte growth factor-mediated inhibition of oxidant-induced cell death

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family (also known as FGF-7), is an important protective factor for epithelial cells. The receptor for KGF (also called FGFR2-IIIb), which has intrinsic tyrosine kinase activity, is expressed specifically on epithelial cells and in the lung epithelium. Administration of KGF has been shown to protect the lung from various insults, but the mechanism of protection is not well understood. To understand the mechanism by which KGF exerts protective functions on epithelial cells, we used the yeast two-hybrid assay to identify proteins that interact with the KGF receptor (KGFR). Here we show that the cytoplasmic domain of KGFR interacts with p21-activated protein kinase (PAK) 4, which is a new member of the PAK family. The PAKs are regulated by the Rho-family GTPases Rac and Cdc42. PAK4 is the most divergent member of the PAK family of proteins and may have distinct functions. However, stimuli that regulate PAK4 activity are not known. Our data show that PAK4 can associate with the KGFR, which is dependent on KGFR tyrosine kinase activity. We show that a dominant negative mutant of PAK4 blocks KGF-mediated inhibition of caspase-3 activation in epithelial cells subjected to oxidant stress. Our data demonstrate that PAK4 is an important mediator of the anti-apoptotic effects of KGF on epithelial cells.     

Hic-5/ARA55: a prostate stroma-specific AR coactivator

Growth factors and cytokines mediate communication between the epithelial and stromal compartments of the prostate. In prostate cancer (PCa), changes in the spatial arrangements of the two compartments (i.e. basement membrane invasion), DNA mutations, or cellular dedifferentiation (i.e. myofibroblasts) leads to significant changes in gene expression within both compartments. This results in altered cytokine and/or growth factor signaling in PCa. Recently, a stromal-specific androgen receptor (AR) coactivator, Hic-5/ARA55, has been identified that may play a role in regulating expression of the growth factor and/or cytokine expression in the prostate. Specifically, Hic-5/ARA55 expression influences androgen-induced keratinocyte growth factor (KGF) expression in WPMY-1 prostate stromal cells. Because Hic-5/ARA55's expression is also altered in PCa, it may play a role in the differential cellular signaling events that occur during tumor progression.     

角质细胞生长因子(KGF)研究进展

角质细胞生长因子(KGF)属于成纤维细胞生长因子(FGFs)家族的成员之一,又称FGF一7,最早由Rubin等(1980)从人类胎肺成纤维细胞培养液中分离提纯获得的。人KGF的cDNA编码一含194个氨基酸的单链多肽,其分子量为26-28KD。KGF由各种来源的间质细胞分泌,与肝素有较强的亲和力,其受体KGFR属于蛋白酪氨酸激酶受体家族,目前已知该家族主要包括FGFR1、FGFR2、FGFR3和FGFR4四位成员,KGFR由FGFR2基因编码,为FGFR-2的剪接形式,即FGFR2Ⅲb,其主要分布于上皮细胞,KGF与靶细胞膜上的受体KGFR特异性结合后,促使受体自身磷酸化,从而启动细胞内信号级联反应,进而发挥多种生物学功能:参与组织器官发育、促进细胞增殖及组织损伤修复、减少放化疗引起的副反应,尤其与癌症的发生发展有着密切的联系。该文就KGF的研究进展进行综述。     

Keratinocyte growth factor/fibroblast growth factor-7-regulated cell migration and invasion through activation of NF-kappaB transcription factors

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor kappaB (NF-kappaB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IkappaBalpha (IkappaBalphaS32A,S36A), which blocked NF-kappaB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-kappaB activation and that NF-kappaB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

The endocytic pathway followed by the keratinocyte growth factor receptor

Keratinocyte growth factor (KGF/FGF7) acts specifically on epithelial cells and regulates their proliferation and differentiation. It binds to and activates a receptor tyrosine kinase, the KGF receptor (KGFR), which is a splicing variant of the fibroblast growth factor receptor 2. The endocytic pathway followed by KGF and its receptor was analyzed here using immunofluorescence and confocal microscopy. After 10 min of internalization at 37 degrees C, both KGF and its receptor were localized in early endosomes, and after 30-60 min of endocytosis ligand and receptor were seen to reach perinuclear late endosomes and not the recycling endosomal compartment. Parallel western blot analysis revealed that KGFRs were tyrosine phosphorylated both at early and late steps of internalization, suggesting that KGF and KGFR remain associated in active complexes through the endocytic pathway. Pulse-chase experiments showed that the internalized KGFRs underwent degradation detectable at 1 h of endocytosis at 37 degrees C, indicating that KGFRs are functionally downregulated.     

Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.     

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia. J. Cell. Physiol. 180:53–60, 1999. © 1999 Wiley-Liss, Inc.     

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.     

Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy.

Stroma and the heparin-binding fibroblast growth factor (FGF) family influence normal epithelial cell growth and differentiation in embryonic and adult tissues. The role of stromal cells and the expression of isoforms of the FGF ligand and receptor family were examined during malignant progression of epithelial cells from a differentiated, slowly growing, nonmalignant model rat prostate tumor. In syngeneic hosts, a mixture of stromal and epithelial cells resulted in nonmalignant tumors which were differentiated and slowly growing. In the absence of the stromal cells, epithelial cells progressed to malignant tumors which were independent of the stroma and undifferentiated. The independence of the malignant epithelial cells from stromal cells was accompanied by a switch from exclusive expression of exon IIIb to exclusive expression of exon IIIc in the FGF receptor 2 (FGF-R2) gene. The FGF-R2(IIIb) isoform displays high affinity for stromal cell-derived FGF-7, whereas the FGF-R2(IIIc) isoform does not recognize FGF-7 but has high affinity for the FGF-2 member of the FGF ligand family. The switch from expression of exclusively exon IIIb to exclusively exon IIIc in the resident FGF-R2 gene was followed by activation of the FGF-2 ligand gene, the normally stromal cell FGF-R1 gene, and embryonic FGF-3 and FGF-5 ligand genes in malignant epithelial cells. Multiple autocrine and potentially intracrine ligand-receptor loops resulting from these alterations within the FGF-FGF-R family may underlie the autonomy of malignant tumor cells.     

Keratinocyte growth factor induces gene expression signature associated with suppression of malignant phenotype of cutaneous squamous carcinoma cells

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.