The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells

Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour‐induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans‐acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P₂‐dependent manner. Membrane‐inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans‐acting factors and cis‐elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells.     

Tec‐Kinase‐Mediated Phosphorylation of Fibroblast Growth Factor 2 is Essential for Unconventional Secretion

Fibroblast growth factor 2 (FGF2) is a potent mitogen that is exported from cells by an endoplasmic reticulum (ER)/Golgi‐independent mechanism. Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes, a process that depends on the phosphoinositide phosphatidylinositol 4,5‐biphosphate (PI(4,5)P₂) at the inner leaflet as well as heparan sulfate proteoglycans at the outer leaflet of plasma membranes; however, additional core and regulatory components of the FGF2 export machinery have remained elusive. Here, using a highly effective RNAi screening approach, we discovered Tec kinase as a novel factor involved in unconventional secretion of FGF2. Tec kinase does not affect FGF2 secretion by an indirect mechanism, but rather forms a heterodimeric complex with FGF2 resulting in phosphorylation of FGF2 at tyrosine 82, a post‐translational modification shown to be essential for FGF2 membrane translocation to cell surfaces. Our findings suggest a crucial role for Tec kinase in regulating FGF2 secretion under various physiological conditions and, therefore, provide a new perspective for the development of a novel class of antiangiogenic drugs targeting the formation of the FGF2/Tec complex.     

Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.     

FGF‐2 promotes osteocyte differentiation through increased E11/podoplanin expression

E11/podoplanin is critical in the early stages of osteoblast‐to‐osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF‐2 on E11‐mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast‐like cells and murine primary osteoblasts to FGF‐2 (10 ng/ml) increased E11 mRNA and protein expression (p < 0.05) after 4, 6, and 24 hr. FGF‐2 induced changes in E11 expression were also accompanied by significant (p < 0.01) increases in Phex and Dmp1 (osteocyte markers) expression and decreases in Col1a1, Postn, Bglap, and Alpl (osteoblast markers) expression. Immunofluorescent microscopy revealed that FGF‐2 stimulated E11 expression, facilitated the translocation of E11 toward the cell membrane, and subsequently promoted the formation of osteocyte‐like dendrites in MC3T3 and primary osteoblasts. siRNA knock down of E11 expression achieved >70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF‐2‐related changes in E11 expression and dendrite formation. FGF‐2 strongly activated the ERK signaling pathway in osteoblast‐like cells but inhibition of this pathway did not block the ability of FGF‐2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF‐2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF‐2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P₂-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.     

Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Basic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.     

Translocation of FGF-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism

The entry of exogenous fibroblast growth factor 2 (FGF-2) to the cytosolic/nuclear compartment was studied and compared with the translocation mechanism used by FGF-1. To differentiate between external and endogenous growth factor, we used FGF-2 modified to contain a farnesylation signal, a CaaX-box. Because farnesylation occurs only in the cytosol and nucleoplasm, farnesylation of exogenous FGF-2-CaaX was taken as evidence that the growth factor had translocated across cellular membranes. We found that FGF-2 translocation occurred in endothelial cells and fibroblasts, which express FGF receptors, and that the efficiency of translocation was increased in the presence of heparin. Concomitantly with translocation, the 18-kDa FGF-2 was N-terminally cleaved to yield a 16-kDa form. Translocation of FGF-2 required PI3-kinase activity but not transport through the Golgi apparatus. Inhibition of endosomal acidification did not prevent translocation, whereas dissipation of the vesicular membrane potential completely blocked it. The data indicate that translocation occurs from intracellular vesicles containing proton pumps and that an electrical potential across the vesicle membrane is required. Translocation of both FGF-1 and FGF-2 occurred during most of G(1) but decreased shortly before the G(1)-->S transition. A common mechanism for FGF-1 and FGF-2 translocation into cells is postulated.     

Secondary structure of cell‐penetrating peptides during interaction with fungal cells

Cell‐penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep‐1, MPG, pVEC, TP‐10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α‐helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep‐1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular‐level interaction with the cell membrane, and these simulations suggested that pVEC, TP‐10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α‐helical conformation. In contrast, penetratin, Pep‐1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.     

The mechanism of coupling between oxido‐reduction and proton translocation in respiratory chain enzymes

The respiratory chain of mitochondria and bacteria is made up of a set of membrane‐associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane‐associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co‐operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X‐ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone‐cytochrome c oxidoreductase and NADH‐ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.     

Fractone‐heparan sulphates mediate FGF‐2 stimulation of cell proliferation in the adult subventricular zone

Objectives: Fractones are extracellular matrix structures that form a niche for neural stem cells and their immediate progeny in the subventricular zone of the lateral ventricle (SVZa), the primary neurogenic zone in the adult brain. We have previously shown that heparan sulphates (HS) associated with fractones bind fibroblast growth factor‐2 (FGF‐2), a powerful mitotic growth factor in the SVZa. Here, our objective was to determine whether the binding of FGF‐2 to fractone‐HS is implicated in the mechanism leading to cell proliferation in the SVZa. Materials and methods: Heparitinase‐1 was intracerebroventricularly injected with FGF‐2 to N‐desulfate HS proteoglycans and determine whether the loss of HS and of FGF‐2 binding to fractones modifies FGF‐2 effect on cell proliferation. We also examined in vivo the binding of Alexa‐Fluor‐FGF‐2 in relationship with the location of HS immunoreactivity in the SVZa. Results: Heparatinase‐1 drastically reduced the stimulatory effect of FGF‐2 on cell proliferation in the SVZa. Alexa‐Fluor‐FGF‐2 binding was strictly co‐localized with HS immunoreactivity in fractones and adjacent vascular basement membranes in the SVZa. Conclusions: Our results demonstrate that FGF‐2 requires HS to stimulate cell proliferation in the SVZa and suggest that HS associated with fractones and vascular basement membranes are responsible for activating FGF‐2. Therefore, fractones and vascular basement membranes may function as a HS niche to drive cell proliferation in the adult neurogenic zone.     

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE‐choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two‐step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell‐extracellular matrix and/or cell–cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

FGF‐8b induces growth and rich vascularization in an orthotopic PC‐3 model of prostate cancer

Fibroblast growth factor 8 (FGF‐8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF‐8b on proliferation of PC‐3 prostate cancer cells and growth of PC‐3 tumors, and to identify FGF‐8b‐associated molecular targets. Expression of ectopic FGF‐8b in PC‐3 cells caused a 1.5‐fold increase in cell proliferation in vitro and a four‐ to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF‐8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT‐qPCR of FGF‐8b mRNA. Microarray analyses revealed significantly altered, up‐ and downregulated, genes in PC‐3 cell cultures (169 genes) and in orthotopic PC‐3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell‐to‐cell signaling and energy production, and the downregulated genes associated with differentiation (DKK‐1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT‐qPCR. In conclusion, our results demonstrate that FGF‐8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF‐8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. 107: 769–784, 2009. © 2009 Wiley‐Liss, Inc.     

Interaction between ERK and GSK3beta mediates basic fibroblast growth factor-induced apoptosis in SK-N-MC neuroblastoma cells

The Ewing's sarcoma family of tumors (ESFT) includes Ewing's sarcoma (ES), Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor. Basic fibroblast growth factor (FGF2) suppresses the growth of ESFT cells and causes their apoptosis. The underlying mechanism is unclear. Using a human peripheral primitive neuroectodermal tumor cell line, SK-N-MC, we demonstrated FGF2 stimulated phosphorylation of ERK1 and ERK2 (pERK1/2) and GSK3beta (pGSK3beta(Tyr-216)), all of which were primarily retained in the cytoplasm. FGF2 promoted the association between ERK and pGSK3beta(Tyr-216). Inhibitors for GSK3beta (TDZD and LiCl) and ERK (PD98059) protected cells from FGF2-induced apoptosis. On the other hand, inhibitors of GSK3beta, but not PD98059 decreased ERK/pGSK3beta(Tyr-216) association and caused a nuclear translocation of pERK1/2. Similarly, expression of a kinase-deficient (K85R) GSK3beta or GSK3beta-small interfering RNA inhibited FGF2-regulated ERK/pGSK3beta(Tyr-216) association and translocated pERK to the nucleus. Both K85R GSK3beta and small interfering RNA offered protection against FGF2-induced cell death. In contrast, overexpression of wild-type GSK3beta sensitized cells to FGF2 cytotoxicity. Hydrogen peroxide and ethanol enhanced FGF2-stimulated pGSK3beta(Tyr-216), ERK/pGSK3beta(Tyr-216) association, and cytoplasmic retention of pERK1/2. As a result, they potentiated FGF2-induced cell death. Taken together, our results suggested that FGF2-induced accumulation of pERK1/2 in the cytoplasm is toxic for SK-N-MC cells. The formation of an ERK.GSK3beta complex retained pERK1/2 in the cytoplasm. In contrast, disruption of the ERK.GSK3beta complex resulted in nuclear translocation of pERK1/2 and offered protection.     

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011. © 2010 Wiley‐Liss, Inc.     

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐positive cells both in inactive and feeding animals. The DA‐induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto‐c (cytochrome c) translocation into the cytosol and methyl‐β‐cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto‐c was under the control of Bcl‐2 and Bad. DA also increased the active caspase‐3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter‐mediated pathway that regulates the PCD of secretory cells in a mitochondrial‐caspase‐dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto‐c, ceramide, Bcl‐2 proteins and caspase‐3, but not caspase‐8, was demonstrated in cells involved in the DA‐induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.     

Fibroblast growth factor 2 protects against renal ischaemia/reperfusion injury by attenuating mitochondrial damage and proinflammatory signalling

Ischaemia‐reperfusion injury (I/RI) is a common cause of acute kidney injury (AKI). The molecular basis underlying I/RI‐induced renal pathogenesis and measures to prevent or reverse this pathologic process remains to be resolved. Basic fibroblast growth factor (FGF2) is reported to have protective roles of myocardial infarction as well as in several other I/R related disorders. Herein we present evidence that FGF2 exhibits robust protective effect against renal histological and functional damages in a rat I/RI model. FGF2 treatment greatly alleviated I/R‐induced acute renal dysfunction and largely blunted I/R‐induced elevation in serum creatinine and blood urea nitrogen, and also the number of TUNEL‐positive tubular cells in the kidney. Mechanistically, FGF2 substantially ameliorated renal I/RI by mitigating several mitochondria damaging parameters including pro‐apoptotic alteration of Bcl2/Bax expression, caspase‐3 activation, loss of mitochondrial membrane potential and K_ATP channel integrity. Of note, the protective effect of FGF2 was significantly compromised by the K_ATP channel blocker 5‐HD. Interestingly, I/RI alone resulted in mild activation of FGFR, whereas FGF2 treatment led to more robust receptor activation. More significantly, post‐I/RI administration of FGF2 also exhibited robust protection against I/RI by reducing cell apoptosis, inhibiting the release of damage‐associated molecular pattern molecule HMBG1 and activation of its downstream inflammatory cytokines such as IL‐1α, IL‐6 and TNF α. Taken together, our data suggest that FGF2 offers effective protection against I/RI and improves animal survival by attenuating mitochondrial damage and HMGB1‐mediated inflammatory response. Therefore, FGF2 has the potential to be used for the prevention and treatment of I/RI‐induced AKI.     

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF‐2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin‐binding growth factor basic fibroblast growth factor (FGF‐2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF‐2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF‐2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF‐2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c‐fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF‐2, and augments and sustains FGF‐2 mediated increases in c‐fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF‐2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF‐2. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 261–277, 2003