Cell type-dependent internalization of the Escherichia coli STb enterotoxin

Previous studies have suggested that internalization of the Escherichia coli STb enterotoxin in human and rat intestinal epithelial cells is involved in STb pathogenesis, but toxin uptake in porcine jejunum epithelium, the in vivo target tissue, still remains elusive. Using flow cytometry, we studied the internalization of fluorescein isothiocyanate-labelled STb in porcine intestinal epithelial IPEC-J2 and murine fibroblast NIH-3T3 cell lines. In contrast to the selective pronase resistance of STb in NIH-3T3 cells at 37 °C, but not at 4 °C, indicative of toxin internalization, most of the toxin was pronase-sensitive at both temperatures in IPEC-J2 cells, indicating reduced uptake, but significant cell surface binding. Actin reorganization is required for STb internalization by NIH-3T3 cells, confirming STb endocytosis in these cells. The toxin receptor, sulfatide, could not explain these internalization differences because both cell lines possessed surface sulfatide and internalized antisulfatide antibodies over time at 37 °C. Inhibition of lipid rafts endocytosis, known to contain sulfatide, with methyl-β-cyclodextrin or genistein, did not influence toxin uptake by either cell line. STb internalization is therefore differentially regulated depending on the cell type, possibly by factors other than sulfatide. Although a small STb fraction could be internalized by porcine intestinal epithelial cells, our findings suggest the ability of STb to induce, from the cell surface, intracellular signalling leading to fluid secretion in porcine intestinal epithelium.     

Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of beta(1)-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, beta(1)-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent beta(1)-integrin internalization. However, beta(1)-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized beta(1)-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased beta(1)-integrin endocytosis. Our data suggest that, in migrating IEC, beta(1)-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.     

Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.     

FGF mediated Sulf1 regulation

FGF signalling is critical for normal embryonic development. Sulf1 has been shown to inhibit FGF activity. The role of FGF4 in Sulf1 regulation was investigated during digital development of the quail autopod. Implantation of FGF4 beads in both the interdigit and at the tip of digit III differentially up-regulated Sulf1 as also confirmed in micromass cultures. FGF4 inhibited interdigital mesodermal apoptosis in a concentration dependent manner. The FGF inhibitor, SU5402, inhibited Sulf1 expression when placed in the interdigital mesoderm. However, when placed at the digital tip, SU5402 induced an ectopic domain of Sulf1 expression and inhibited further phalange formation.     

Protective effects of activated vitamin D receptor on radiation-induced intestinal injury

Radiation-induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) -induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti-injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation-induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation-induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation-induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation

FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.     

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.     

RAGE: A potential therapeutic target during FGF1 treatment of diabetes-mediated liver injury

As a serious metabolic disease, diabetes causes series of complications that seriously endanger human health. The liver is a key organ for metabolizing glucose and lipids, which substantially contributes to the development of insulin resistance and type 2 diabetes mellitus (T2DM). Exogenous fibroblast growth factor 1 (FGF1) has a great potential for the treatment of diabetes. Receptor of advanced glycation end products (RAGE) is a receptor for advanced glycation end products that involved in the development of diabetes-triggered complications. Previous study has demonstrated that FGF1 significantly ameliorates diabetes-mediated liver damage (DMLD). However, whether RAGE is involved in this process is still unknown. In this study, we intraperitoneally injected db/db mice with 0.5 mg/kg FGF1. We confirmed that FGF1 treatment not only significantly ameliorates diabetes-induced elevated apoptosis in the liver, but also attenuates diabetes-induced inflammation, then contributes to ameliorate liver dysfunction. Moreover, we found that diabetes triggers the elevated RAGE in hepatocytes, and FGF1 treatment blocks it, suggesting that RAGE may be a key target during FGF1 treatment of diabetes-induced liver injury. Thus, we further confirmed the role of RAGE in FGF1 treatment of AML12 cells under high glucose condition. We found that D-ribose, a RAGE agonist, reverses the protective role of FGF1 in AML12 cells. These findings suggest that FGF1 ameliorates diabetes-induced hepatocyte apoptosis and elevated inflammation via suppressing RAGE pathway. These results suggest that RAGE may be a potential therapeutic target for the treatment of DMLD.     

Duration of ERK1/2 phosphorylation induced by FGF or ocular media determines lens cell fate

The ocular environment is important for the establishment and maintenance of lens growth patterns and polarity. In the anterior chamber of the eye, the aqueous humour regulates lens epithelial cell proliferation whereas in the posterior, the vitreous humour regulates the differentiation of the lens cells into fiber cells. Members of the fibroblast growth factor (FGF) growth factor family have been shown to induce lens epithelial cells to undergo cell division and differentiate into fibers, with a low dose of FGF able to induce cell proliferation (but not fiber differentiation), and higher doses required to induce fiber differentiation. Both these cellular events have been shown to be regulated by the MAPK/ERK1/2 signalling pathway. In the present study, to better understand the contribution of ERK1/2 signalling in regulating lens cell proliferation and differentiation, we characterized the ERK1/2 signalling profiles induced by different doses of FGF, and compared these to those induced by the different ocular media. Here, we show that FGF induced a dose-dependent sustained activation of ERK1/2, with both a high (fiber differentiating) dose of FGF and vitreous, stimulating and maintaining a prolonged (up to 18 hr) ERK1/2 phosphorylation profile. In contrast, a lower (proliferating) dose of FGF, and aqueous, stimulated ERK1/2 phosphorylation for only up to 6 hr. If we selectively reduce the 18 hr ERK1/2 phosphorylation profile induced by vitreous to 6 hr, by specifically blocking FGF receptor signalling, the vitreous now fails to induce lens fiber differentiation but retains the ability to induce lens cell proliferation. These findings not only provide insights into the important role that FGF plays in the different ocular media that bathe the lens, but enlighten us on some of the putative molecular mechanisms by which one specific growth factor, in this case FGF, can elicit a different cellular response in the same cell type.     

Autocrine FGF signaling is required for vascular smooth muscle cell survival in vitro

Expression of both basic fibroblast growth factor (bFGF) and FGF receptors (FGFR) by vascular smooth muscle cells suggests that autocrine FGF signaling mechanisms may have important functions. Inhibition of smooth muscle cell bFGF expression provokes apoptosis, suggesting that endogenous bFGF generates an anti-apoptotic signal. The purpose of this study was to determine whether the survival function of endogenous bFGF requires signaling through FGFR. A recombinant adenovirus encoding a truncated murine FGFR-1 lacking the kinase domain (DN-FGFR) efficiently expressed the transgene in cultured rat aortic smooth muscle cells. The truncated receptor acted in a dominant negative fashion to effectively prevent receptor-mediated signaling, assessed by phosphorylation of p42/p44 MAP kinase. Expression of DN-FGFR provoked apoptosis of SMC in a dose-dependent fashion that was insensitive to recombinant bFGF but could be rescued by platelet derived growth factor or epidermal growth factor. Heterologous growth factor rescue was inhibited by PD98059, an inhibitor of MEK (MAP kinase-kinase). These data demonstrate that inhibition of FGF receptor activation results in apoptosis and suggest that an intact autocrine FGF signaling loop is required for vascular smooth muscle cell survival in vitro. These findings also implicate the Ras/Raf/MEK /MAP kinase cascade in generating or sustaining the survival signal. The functional significance of an autocrine FGF signaling loop in non-transformed cells has important implications for cardiovascular development, remodeling and disease. J. Cell. Physiol. 177:58–67, 1998. © 1998 Wiley-Liss, Inc.     

Phosphorylation of fibroblast growth factor (FGF) receptor 1 at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus

Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the alpha isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38alpha. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38alpha in a cell-free system. These data demonstrate a crucial role for p38alpha MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38alpha MAPK-mediated serine phosphorylation of FGFR1.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.     

FGF信号通路在内耳发育调控和毛细胞再生中的作用

内耳是感受听觉和平衡觉的复杂器官。在内耳发育过程中,成纤维生长因子(fibroblast growth factor, FGF)信号通路参与了听基板的诱导、螺旋神经节(statoacoustic ganglion, SAG)的发育以及Corti器感觉上皮的分化。FGF信号开启了内耳早期发育的基因调控网络,诱导前基板区域以及听基板的形成。正常表达的FGF信号分子可促进听囊腹侧成神经细胞的特化,但成熟SAG神经元释放的过量FGF5可抑制此过程,形成负反馈环路使SAG在稳定状态下发育。FGF20在Notch信号通路的调控下参与了前感觉上皮区域向毛细胞和支持细胞的分化过程,而内毛细胞分泌的FGF8可调控局部支持细胞分化为柱细胞。人类FGF信号通路异常可导致多种耳聋相关遗传病。此外,FGF信号通路在低等脊椎动物毛细胞自发再生以及干细胞向内耳毛细胞诱导过程中都起到了关键作用。本文综述了FGF信号通路在内耳发育调控以及毛细胞再生中的作用及其相关研究进展,以期为毛细胞再生中FGF信号通路调控机制的阐明奠定理论基础。     

Intracellular signalling pathways involved in mesoderm induction by FGF.

We have examined the possible role of two signal transducing mechanisms, tyrosine phosphorylation and activation of protein kinase C (PKC), during fibroblast growth factor (FGF)-induced mesoderm induction in Xenopus. Tyrosine phosphorylation was examined through the use of a monoclonal anti-phosphotyrosine antibody. This antibody was shown to recognize the FGF receptor crosslinked to radioiodinated FGF. We also studied the response of Xenopus ectodermal explants to sodium orthovanadate, a compound that has been shown to elevate intracellular phosphotyrosine levels. Thirty percent of explants cultured in 100 microM vanadate were induced. In addition, vanadate synergized with FGF to give inductions that were more dorsal in nature than either vanadate or FGF alone. The role of PKC was evaluated by measuring PKC activity during mesoderm induction by FGF and by examining the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on explants. TPA did not induce mesoderm, however, activation of PKC was detected in FGF-treated explants. Therefore, activation of the PKC pathway alone is not sufficient for mesoderm induction. Simultaneous treatment with TPA and FGF resulted in a significant inhibition of mesoderm induction by FGF, suggesting that activation of PKC could be part of a negative feedback mechanism. In contrast, TPA had no effect on induction by activin A.     

Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells

Platelet-activating factor (PAF) is a phospholipid inter- and intracellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathological effects on intestinal epithelial cells (IEC). PAF induces Cl(-) channel activation, which is associated with intracellular acidosis and apoptosis. Using the rat small IEC line IEC-6, electrophysiological experiments demonstrated that PAF induces Cl(-) channel activation. This PAF-activated Cl(-) current was inhibited by Ca(2+) chelation and a calcium calmodulin kinase II inhibitor, suggesting PAF activation of a Ca(2+)-activated Cl(-) channel. To determine the pathological consequences of Cl(-) channel activation, microfluorimetry experiments were performed, which revealed PAF-induced intracellular acidosis, which is also inhibited by the Cl(-) channel inhibitor 4,4'diisothiocyanostilbene-2,2'disulfonic acid and Ca(2+) chelation. PAF-induced intracellular acidosis is associated with caspase 3 activation and DNA fragmentation. PAF-induced caspase activation was abolished in cells transfected with a pH compensatory Na/H exchanger construct to enhance H(+) extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl(-) channel dependent on both Ca(2+) and calcium calmodulin kinase II phosphorylation, we generated ClC-3 knockdown cells using short hairpin RNA. PAF induced Cl(-) current; acidosis and apoptosis were all significantly decreased in ClC-3 knockdown cells. Our data suggest a novel mechanism of PAF-induced injury by which PAF induces intracellular acidosis via activation of the Ca(2+)-dependent Cl(-) channel ClC-3, resulting in apoptosis of IEC.