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。     

Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.     

Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor

Basic fibroblast growth factor (FGF)-2 is important for vessel formation and/or maintenance of vascular integrity in the embryo. FGF signaling may be mediated through transmembrane tyrosine kinase receptors or directly through intracellular pathways that do not involve receptor activation. To determine the role of receptor-mediated signaling in endothelial cells, an adenovirus encoding truncated FGF receptor (FGFR)-1, under the control of the cytomegalovirus promoter, was expressed in endothelial cells. FGF signaling was impaired, as indicated by inhibition of MAPK phosphorylation. Functional consequences included inhibition of endothelial cell migration and induction of apoptosis. To address the role of endothelial FGFR signaling in vascular development, recombinant adenovirus encoding a dominant-negative FGFR was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Previous studies demonstrated that transgenes delivered via adenovirus, under the control of the cytomegalovirus promoter, are expressed selectively in the developing vasculature. Embryos expressing a control adenovirus developed normally, whereas those expressing the FGFR-1 mutant exhibited abnormal embryonic and extra-embryonic vascular development. These data demonstrate that FGF, by signaling through the FGFR, plays a pivotal role in the development and maintenance of a mature vascular network in the embryo.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

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.     

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.     

Novel phosphotyrosine targets of FGFR2IIIb signaling

In partnership exclusively with the epithelial FGFR2IIIb isotype and a structurally-specific heparan sulfate motif, stromal-derived FGF7 delivers both growth-promoting and growth-limiting differentiation signals to epithelial cells that promote cellular homeostasis between stromal and epithelial compartments. Intercompartmental homeostasis supported by FGF7/FGFR2IIIb is subverted in many solid epithelial tumors. The normally mesenchymal-derived homologue FGFR1 drives proliferation and a progressive tumor-associated phenotype when it appears ectopically in epithelial cells. In order to understand the mechanism underlying the unique biological effects of FGFR2IIIb, we developed an inducible FGFR2IIIb expression system that is specifically dependent on FGF7 for activation in an initially unresponsive cell line to avoid selection for only the growth-promoting aspects of FGFR2IIIb signaling. We then determined FGF7/FGFR2IIIb signaling-specific tyrosine phosphorylated proteins within 5 min after FGF7 stimulation by phosphopeptide immunoaffinity purification and nano-LC-MS/MS. The FGF7/FGFR2 pair caused tyrosine phosphorylation of multiple proteins that have been implicated in the growth stimulating activities of FGFR1 that included multi-substrate organizers FRS2α and IRS4, ERK2 and phosphatases SHP2 and SHIP2. It uniquely phosphorylated CDK2 and phosphatase PTPN18 on sites involved in the attenuation of cell proliferation, and several factors that maintain nuclear-cytosolic relationships (emerin and LAP2), protein structure and other cellular fine structures as well as some proteins of unknown functions. Several of the FGF7/FGFR2IIIb-specific targets have been associated with maintenance of function and tumor suppression and disruption in tumors. In contrast, a number of pTyr substrates associated with FGF2/FGFR1 that are generally associated with intracellular Ca²⁺-phospholipid signaling, membrane and cytoskeletal plasticity, cell adhesion, migration and the tumorigenic phenotype were not observed with FGF7/FGFR2IIIb. Our findings provide specific downstream targets for dissection of causal relationships underlying the distinct role of FGF7/FGFR2IIIb signaling in epithelial cell homeostasis.     

Differential signal transduction of alternatively spliced FGFR2 variants expressed in human mammary epithelial cells

Gene amplification and protein overexpression of fibroblast growth factor receptor 2 (FGFR2) characterize the SUM-52 breast cancer cell line developed in our laboratory. SUM-52 cells express nine distinct alternatively spliced isoforms of FGFR2. Among these isoforms are two otherwise identical FGFR2 variants that express either the C1 or C3 carboxyl terminus. FGFR2-C3 variants are not normally expressed by human mammary epithelial (HME) cells, and we have shown that overexpression of FGFR2-C3 in HME cells results in potent transformation. In particular, FGFR2-C3 expression leads to robust levels of constitutively tyrosine phosphorylated FRS2 in the absence of ligand stimulation. In contrast, overexpressed FGFR2-C1 requires constant stimulation with exogenous keratinocyte growth factor (KGF) to mimic the signaling capability of FGFR2-C3. However, activation of FRS2 that results from KGF-stimulated FGFR2-C1 signaling is transient and is associated with a mobility shift of FRS2 not observed when this signaling molecule is activated by the C3 isoform of FGFR2. Mutation of the only tyrosine phosphorylated site present in the C1 terminus and absent from C3, Tyr769, did not yield a receptor that rivaled the potent signaling of FGFR2-C3. We therefore conclude that aberrant expression of alternatively spliced isoforms of FGFR2 with the C3 carboxyl terminus in the SUM-52 breast cancer cells results in sustained activation of signal transduction leading to transformation.     

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET² signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser–Ser–Ser) and YGS (Tyr–Gly–Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.     

FGFR1 is essential for N-acetyl-seryl-aspartyl-lysyl-proline regulation of mitochondrial dynamics by upregulating microRNA let-7b-5p

Fibroblast growth factor receptor (FGFR) 1 plays a key role in endothelial homeostasis by inducing microRNA (miR) let-7. Our previous paper showed that anti-fibrotic effects of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) were associated with restoring diabetes-suppressed expression of FGFR1 and miR let-7, the key contributor of mitochondrial biogenesis, which is regulated by mitochondrial membrane GTPase proteins (MFN2 and OPA1). Here, we found that the FGFR1 signaling pathway was critical for AcSDKP in maintaining endothelial mitochondrial biogenesis through induction of miR let-7b-5p. In endothelial cells, AcSDKP restored the triple cytokines (TGF-β2, interleukin-1β, tumor necrosis factor-α)-suppressed miR let-7b-5p and protein levels of the mitochondrial membrane GTPase. This effect of AcSDKP was lost with either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1-treated cells. Similarly, AcSDKP had no effect on the miR let-7b-5p inhibitor-suppressed GTPase levels in endothelial cells. In addition, a miR let-7b-5p mimic restored the levels of FRS2 siRNA-reduced GTPases in endothelial cells. These findings were also confirmed using MitoTracker Green and an immunofluorescence assay. Our results demonstrated that the AcSDKP-FGFR1 signaling pathway is critical for maintaining mitochondrial dynamics by control of miR let-7b-5p in endothelial cells.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

Fibroblast growth factor-2 promotes catabolism via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.     

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.     

Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells

The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.     

FGFR3-expressing smooth muscle-like stromal cells differentiate in response to FGFR2IIIb-expressing prostate tumor cells and delay tumor progression

Evolution of unresponsiveness to homeostasis-promoting signals from the microenvironment is a hallmark of malignant tumor cells. In Dunning R3327 model rat prostate tumors that are comprised of distinct stromal and epithelial compartments, progression from non-malignant, androgen-responsive tumors to malignancy is characterized by loss of compartmentation coincident with a loss of resident epithelial cell FGFR2IIIb that receives instructive signals from stromal FGF7 and FGF10. Restoration of FGFR2IIIb to malignant tumor cells restores responsiveness to stromal cells, restores distinct stromal and epithelial compartments, and retards malignant progression. Cultured stromal cells from two-compartment tumors are comprised of smooth muscle α-actin-positive cells that express predominantly FGFR3 and fibroblast-like cells devoid of α-actin and FGFR3. Here, we show that it is primarily the smooth muscle cell-like α-actin-expressing stromal cells that survive, morphologically differentiate, and delay tumor incidence and size in the presence of malignant cells in which FGFR2IIIb has been restored. Expression of FGFR3 by transfection in the fibroblast-like stromal cells conferred ability to respond similar to the smooth muscle cell-like stromal cells in which FGFR3 is normally resident. These results highlight the importance of the two-way communication back and forth between stroma and epithelium that is mediated by signaling within the FGFR family during progression to malignancy.     

细胞质内微环境直接影响FGFR1酪氨酸激酶介导的SNT1细胞特异性磷酸化

成纤维细胞生长因子受体(FGFR)介导的SNT1(亦称为FRS2)底物磷酸化具有宿主细胞以及受体特异性。为探明这种宿主细胞特异性的决定因素,我们构建了1个FGFR2Ⅲb/R1嵌合受体。该嵌合受体具有1个FGFR2Ⅲb的胞外片段及1个FGFR1蛋白质酪氨酸激酶片断。当表达在3T3细胞(内源性受体为FGFR1并能强烈响应FGFR1的信号)以及DTE-R1/100细胞时,该嵌合受体能即刻诱导SNT1磷酸化。DTE-R1/100细胞为经长期培养的带有外源性FGFR1的非恶性前列腺肿瘤上皮细胞(DTE)并已获得未转化DTE细胞所不具备的FGFR1信号响应性。与此相反,当表达在非转化DTE细胞或未经长期培养的FGFR1转化细胞(DTE-R1)时,FGFR2Ⅲb/R1嵌合受体则无法诱导SNT1磷酸化。我们曾报导DTE细胞对FGFR1介导的SNT1磷酸化活力及其刺激细胞生长信号的响应性是一种获得性的性质,这种性质的获得与细胞恶化是紧密联系在一起的。在此我们进一步证明FGFR介导的SNT1磷酸化具有宿主细胞特异性。这些结果表明细胞内围绕着激酶的微环境而不是细胞外环境决定了SNT1是否可为FGFR1所磷酸化。而且,长期受外源性FGFR1刺激诱发DTE细胞内微环境的变化,从而使表达在DTE细胞里的FGFR1激酶可强烈地磷酸化SNT1。     

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.