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.     

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19?cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation.     

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.     

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.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

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.     

Polarized endocytosis of the keratinocyte growth factor receptor in migrating cells: role of SRC-signaling and cortactin

Cell migration is a physiological process that requires endocytic trafficking and polarization of adhesion molecules and receptor tyrosine kinases (RTKs) to the leading edge. Many growth factors are able to induce motility by binding to specific RTK on target cells. Among them, keratinocyte growth factor (KGF or FGF7) and fibroblast growth factor 10 (FGF10), members of the FGF family, are motogenic for keratinocytes, and exert their action by binding to the keratinocyte growth factor receptor (KGFR), a splicing variant of FGFR2, exclusively expressed on epithelial cells. Here we analyzed the possible role of cortactin, an F-actin binding protein which is tyrosine phosphorylated by Src and is involved in KGFR-mediated cell migration, in the KGFR endocytosis and polarization to the leading edge of migrating cells upon ligand-induced stimulation. Biochemical phosphorylation study revealed that both KGF and FGF10 were able to induce tyrosine phosphorylation of Src and in turn of cortactin, as demonstrated by using the specific pharmacological Src-inhibitor SU6656, although FGF10 effect was delayed with respect to that promoted by KGF. Immunofluorescence analysis demonstrated the polarized localization of KGFR upon ligand stimulation to the leading edge of migrating keratinocytes, process that was regulated by Src. Moreover, we showed that the colocalization of cortactin with KGFR at the plasma membrane protrusions and on early endosomes after KGF and FGF10 treatment was Src-dependent. Further, by using a RNA interference approach through microinjection, we showed that cortactin is required for KGFR endocytosis and that the clathrin-dependent internalization of the receptor is a critical event for its polarization. Finally, KGFR expression and polarization enhanced cell migration in a scratch assay. Our results indicate that both Src and cortactin play a key role in the KGFR endocytosis and polarization at the leading edge of migrating keratinocytes, supporting the crucial involvement of RTK trafficking in cell motility.     

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.     

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.     

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。     

Non-genomic effects of the androgen receptor and vitamin D agonist are involved in suppressing invasive phenotype of prostate cancer cells

Suppression of invasive phenotype is essential in developing new therapeutic tools to treat prostate cancer (PC). Evidence indicates that androgen-dependent (AD) prostate cancer cells are characterized by a lower malignant phenotype. We have demonstrated that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion of these cells through modulation of alpha6beta4 integrin expression, indicating a genotropic effect of androgens in inhibiting invasion ability of AD PC cells. Later on, we have shown that also a non-genotropic mechanism is involved in such an effect. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3-AR cells co-localizes with the EGFR receptors (EGFR) in PC3-AR cells. Co-immunoprecipitation studies both in PC3-AR cells and in the AD cell line LNCaP that physiologically express both receptors, confirm the occurrence of an interaction between of the two proteins. In PC3-AR cells, we demonstrated a disruption of EGFR signalling properties (reduced EGF-induced EGFR autotransphosphorylation, reduced EGF-stimulated PI3K activity as well as EGFR-PI3K interaction) contributing to the lower invasive phenotype of these cells. In another study, we investigated the effects of a new Vitamin D analogue, BXL628, on invasion in response to KGF in the androgen-independent PC cell line DU145. We found that the compound was able to reduce proliferation and invasion of the cells in response to the growth factor. In addition, we found that KGF-induced autotransphosphorylation of KGF receptor (KGFR) and PI3K activation were suppressed after short-term (5min) pre-treatment with the analogue before addition of KGF. Collectively, these studies demonstrate that a non-genotropic effect due to a direct interaction of the androgen receptor with EGFR and to a rapid effect of a Vitamin D agonist on KGFR may disrupt signalling of GF leading to decreased tumorigenicity and a less malignant phenotype of PC cells in vitro.     

Differential regulated expression of keratinocyte growth factor and its receptor in experimental and human liver fibrosis

BACKGROUND AND AIM: Immunomodulatory and protective properties have been identified for the keratinocyte growth factor (KGF). For hepatocytes, pro-proliferative and anti-apoptotic effects of this growth factor have been reported in vitro. This study was designed to characterize a putative role of KGF in observed histomorphological changes in both, human and experimental liver fibrosis. METHODS: Liver fibrosis and cirrhosis was induced in rats by repetitive exposure to phenobarbitone and increasing doses of carbon tetrachloride. Human samples were obtained from patients undergoing surgery for partial hepatectomy or transplantation. Organ samples were scored for inflammation and morphological changes. Expression of KGF and its receptor (KGFR) mRNA was quantified by real-time RT-PCR. Protein expression and receptor phosphorylation was determined by Western blot analysis. In-situ hybridization and immunohistochemistry were utilized to determine distribution of KGF and KGFR in the liver. RESULTS: Expression of KGF was significantly increased in damaged liver tissue in correlation to the degree of fibrosis, whereas expression of the receptor was up-regulated in early stages of liver fibrosis and down-regulated in cirrhotic organs. Protein expression of this growth factor and its receptor correlated with the alterations in mRNA. KGF expression was restricted to mesenchymal cells, whereas expression of KGFR was detected on hepatocytes only. CONCLUSION: The expression of KGF and KGFR is differentially and significantly regulated in damaged liver tissue. This growth factor might therefore not only contribute to morphological alterations but also regeneration of liver parenchyma most likely mediated by indirect mechanisms of action.     

角质细胞生长因子研究进展

角质细胞生长因子(KGF)从属于成纤维细胞生长因子家族。KGF基因表达受多种细胞因子调控。KGF与受体KGFR特异性的结合发挥其多种生物学功能：参与组织、器官的发育;参与皮肤、胃、肠、肾、膀胱、肺等上皮的损伤修复;减少放、化疗所带来的副作用,具有损伤防护功能;KGF与肿瘤密不可分。     

Keratinocyte growth factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 lung epithelial cells

Acute respiratory distress syndrome (ARDS) is a syndrome characterized by the rapid influx of protein-rich edema fluid into the air spaces. The magnitude of alveolar epithelial cell injury is a key determinant of disease severity and an important predictor of patient outcome. The alveolar epithelium is positioned at the interface of the host response in the initiation, progression, and recovery phase of the disease. Keratinocyte growth factor (KGF) is a potent survival factor unique to the epithelium that promotes lung epithelial cell survival, accelerates wound closure, and reduces fibrosis. We therefore hypothesized that KGF preserves lung function by inhibiting apoptosis through activation of a signal transduction pathway responsible for cell survival. To test this hypothesis we determined that KGF inhibits death following Fas activation, a relevant apoptosis pathway, and then determined that cell survival is mediated through activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt kinase signal transduction pathway. We found that KGF induces a dose- and time-dependent increase in Akt kinase activity and that, as expected, activation of Akt via KGF is PI3K dependent. KGF inhibited Fas-induced apoptosis as measured by a reduction in apoptotic cells and caspase-3 activity. This investigation supports our original hypothesis that KGF protects the lung epithelium by inhibiting apoptosis and that protection occurs through activation of PI3K/Akt-mediated cell survival pathway. Our results are in agreement with other reports that identify the PI3K/Akt axis as a key intracellular pathway in the lung epithelium that may serve as a therapeutic target to preserve epithelial integrity during inflammation.     

角质细胞生长因子的研究进展

角质细胞生长因子(KGF)是成纤维细胞生长因子(FGFs)家族的成员,即FGF-7,最初是从人胚胎肺成纤维细胞的培养上清中分离纯化获得的。成熟KGF为一163个氨基酸残基的单链多肽,分子量为26—28KD。KGF由各种来源的间质细胞分泌,受体分布于上皮细胞,其生物学活性是特异性地促进上皮细胞的增殖、迁移和分化。KGF的表达受激素和一些细胞因子的调控。有关研究表明,KGF对肺泡Ⅱ型细胞的增殖以及皮肤、胃肠道粘膜和角膜损伤的修复具有十分重要的作用。     

Pattern of expression of the KGF receptor and its ligands KGF and FGF-10 during postnatal mouse mammary gland development

The expression of the KGF receptor (KGFR) and its stromal ligands, KGF and FGF-10, was compared during mouse mammary gland development. KGFR expression in mammary parenchyma is maximal in mature virgin mice, declines during pregnancy and lactation, but rises after weaning. The rise in KGFR mRNA in the virgin animal corresponds to parenchymal growth. The fall in KGFR expression in pregnancy is driven by hormone-induced alveolar differentiation since the level of KGFR mRNA is 5-fold higher in isolated ductal cells compared to alveolar cells. KGF and FGF-10 expression patterns differ during ductal development. FGF-10 is also expressed at about a 15-fold higher molar level than KGF. During pregnancy and lactation, expression of KGF and FGF-10 decreases in intact fat pads but is unchanged in parenchyma-free fat pads. Thus, the decrease in KGF and FGF-10 expression observed in intact glands during pregnancy and lactation is not a direct consequence of the changing hormonal milieu but more likely reflects an increase in the ratio of epithelium to stroma. Differences in the level and pattern of expression of mRNA for KGF, FGF-10, and the KGFR during postnatal development of the mouse mammary gland are a result of morphological development, changes in the ratio of stroma to epithelium, and hormonal regulation of cell differentiation. These changes suggest that the biological roles that these growth factors play are regulated by fluctuations in both growth factor and growth factor receptor expression and that KGF and FGF-10 may have different regulatory functions.     

AKT and MAPK signaling in KGF-treated and UVB-exposed human epidermal cells

Regulation of proliferation and differentiation in keratinocyte is a complex and dynamic process that involves activation of multiple signaling pathways triggered by different growth factors. Keratinocyte growth factor (KGF) is not only a potent mitogen, but differently from other growth factors, is a potent inducer of differentiation. The MAP kinase and AKT pathways are involved in proliferation and differentiation of many cell types including keratinocytes. We investigated here the role of KGF in modulating AKT and MAPK activity during differentiation of human keratinocytes. Our results show that the mechanisms of action of KGF are dose-dependent and that a sustained activation of the MAPK signaling cascade causes a negative regulation of AKT. We also demostrated increasing expression of KGFR substrates, such as PAK4 during keratinocyte differentiation parallel to the receptor upregulation.