Phosphatidylserine externalization and membrane blebbing are involved in the nonclassical export of FGF1

The mechanisms of nonclassical export of signal peptide-less proteins remain insufficiently understood. Here, we demonstrate that stress-induced unconventional export of FGF1, a potent and ubiquitously expressed mitogenic and proangiogenic protein, is associated with and dependent on the formation of membrane blebs and localized cell surface exposure of phosphatidylserine (PS). In addition, we found that the differentiation of promonocytic cells results in massive FGF1 release, which also correlates with membrane blebbing and exposure of PS. These findings indicate that the externalization of acidic phospholipids could be used as a pharmacological target to regulate the availability of FGF1 in the organism.     

The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export

The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.     

Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.

In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.     

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.     

Cell size increased in tissues from transgenic mice overexpressing a cell surface growth-related and cancer-specific hydroquinone oxidase, tNOX, with protein disulfide-thiol interchange activity

tNOX (ENOX2), a cancer-specific and growth-related cell surface protein with protein disulfide-thiol interchange and hydroquinone (NADH) oxidase activities was overexpressed in a transgenic mouse model. Female transgenic mice grew faster than wild type as did embryonic fibroblast cells prepared from the transgenic mice. The tissue expression of tNOX mRNA was greatest in heart, lung and liver. When these tissues were analyzed for cell size, the cells from the tissues of transgenic animals were, on average, 20% larger in surface area than cells from corresponding wild-type tissues. Also analyzed were cells of intestine, spleen and kidney in which tNOX overexpression was observed but to a lesser extent. Cell size was increased as well with intestine and kidney but less so with spleen. At the end of the study, carcass weights of the transgenic animals were greater than those of wild type. This increase in carcass weight was reflected in an increase in femur weight and thickness in both male and female transgenic mice but not in femur length. Other carcass parameters such as skin weight and body fat or body fluids were unchanged or changes were insufficient to account for the increased carcass weight. The findings are consistent with the property of tNOX observed in studies with cultured cells as contributing to the enlargement phase of cell growth.     

Syndecan-4 modulates basic fibroblast growth factor 2 signaling in vivo

Syndecan-4 is one of the principal heparan sulfate-carrying proteins on the cell surface. Unlike other members of syndecan family, syndecan-4 mediates phosphatidylinositol 4,5-bisphosphate 2 (PIP(2))-dependent PKC-alpha activation, and overexpression of syndecan-4 in vitro results in enhanced FGF2 signaling. The present study was designed to test the functional effect of increased syndecan-4 expression in endothelial cells in transgenic mice. Several transgenic mice lines expressing syndecan-4 cDNA under control of human endothelial nitric oxide (NO) synthase (eNOS) promoter were generated. Exogenous syndecan-4 was mainly expressed in the heart, brain, and lungs. In particular, the heart demonstrated the greatest increase in the ratio of transgenic-to-native syndecan-4 gene expression. Vessels from the eNOS-syndecan-4 mice demonstrated more pronounced vasodilation to FGF2 but not to VEGF-A(165), sodium nitroprusside, and A 23187 compared with wild-type mice. To elucidate the mechanism of this effect, we measured NO release from primary cardiac endothelial cells isolated from transgenic or wild-type adult mice. Cells from the eNOS-syndecan-4 transgenic mice had a significant increase in FGF2- and VEGF-A(165)-induced NO release compared with endothelial cells from the wild-type mice. However, the absolute magnitude of this increase was higher for FGF2 than VEGF-A(165). In conclusion, enhanced syndecan-4 expression in mouse cardiac endothelial cells results in preferential augmentation of FGF2 but not VEGF-A(165)-induced NO release.     

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.     

FGF-18, a Novel Member of the Fibroblast Growth Factor Family, Stimulates Hepatic and Intestinal Proliferation

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.     

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.     

FGF23 ameliorates ischemia-reperfusion induced acute kidney injury via modulation of endothelial progenitor cells: targeting SDF-1/CXCR4 signaling

The levels of fibroblast growth factor 23 (FGF23) rapidly increases after acute kidney injury (AKI). However, the role of FGF23 in AKI is still unclear. Here, we observe that pretreatment with FGF23 protein into ischemia-reperfusion induced AKI mice ameliorates kidney injury by promoting renal tubular regeneration, proliferation, vascular repair, and attenuating tubular damage. In vitro assays demonstrate that SDF-1 induces upregulation of its receptor CXCR4 in endothelial progenitor cells (EPCs) via a non-canonical NF-κB signaling pathway. FGF23 crosstalks with the SDF-1/CXCR4 signaling and abrogates SDF-1-induced EPC senescence and migration, but not angiogenesis, in a Klotho-independent manner. The downregulated pro-angiogenic IL-6, IL-8, and VEGF-A expressions after SDF-1 infusion are rescued after adding FGF23. Diminished therapeutic ability of SDF-1-treated EPCs is counteracted by FGF23 in a SCID mouse in vivo AKI model. Together, these data highlight a revolutionary and important role that FGF23 plays in the nephroprotection of IR-AKI.Subject terms: Extracellular signalling molecules, DNA methylation, Acute kidney injury, Experimental models of disease     

A new pathway for protein export in Saccharomyces cerevisiae

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.     

Adamts1 is highly induced in rachitic bones of FGF23 transgenic mice and participates in degradation of non-mineralized bone matrix collagen

Transgenic mice overexpressing fibroblast growth factor 23 (FGF23) in osteoblasts have a rachitic bone phenotype. These mice display hypomineralized bones, increased expression of osteoblast markers, but osteoclast numbers are unaltered or slightly reduced. Paradoxically, they show increased serum levels of the bone resorption marker CTX, a type I collagen degradation fragment. Here we analyzed a matrix metalloproteinase- (MMP-) like secreted protease, Adamts1, that has previously been associated with osteoblastic type I collagen breakdown in vitro. Bones from FGF23 transgenic (tg) mice displayed increased Adamts1 protein upon both immunohistological staining and Western blotting. We further found Adamts1 protein together with excessively degraded type I collagen in the non-mineralized bone fraction of FGF23 tg mice. A similar degradation pattern of type I collagen was noticed upon forced expression of Adamts1 in osteoblastic cells in vitro. Importantly, these Adamts1-expressing osteoblastic cells exhibited increased release of CTX fragments when cultured on demineralized bone discs. Together, these results demonstrate for the first time that Adamts1 can be highly induced in bone tissue and that this MMP-like protease can increase osteoblastic release of CTX fragments from non-mineralized bone. Thus, Adamts1 potentially contributes to the increased serum levels of CTX in rickets/osteomalacia.     

FGF21 mediates alcohol-induced adipose tissue lipolysis by activation of systemic release of catecholamine in mice

Alcohol consumption leads to adipose tissue lipoatrophy and mobilization of FFAs, which contributes to hepatic fat accumulation in alcoholic liver disease. This study aimed to investigate the role of fibroblast growth factor (FGF)21, a metabolic regulator, in the regulation of chronic-binge alcohol-induced adipose tissue lipolysis. FGF21 KO mice were subjected to chronic-binge alcohol exposure, and epididymal white adipose tissue lipolysis and liver steatosis were investigated. Alcohol exposure caused adipose intracellular cAMP elevation and activation of lipolytic enzymes, leading to FFA mobilization in both WT and FGF21 KO mice. However, alcohol-induced systemic elevation of catecholamine, which is known to be a major player in adipose lipolysis by binding to the β-adrenergic receptor, was markedly inhibited in KO mice. Supplementation with recombinant human FGF21 to alcohol-exposed FGF21 KO mice resulted in an increase in fat loss in parallel with an increase of circulating norepinephrine concentration. Furthermore, alcohol consumption-induced fatty liver was blunted in the KO mice, indicating an inhibition of fatty acid reverse transport from adipose to the liver in the KO mice. Taken together, our studies demonstrate that FGF21 KO mice are protected from alcohol-induced adipose tissue excess-lipolysis through a mechanism involving systemic catecholamine release.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.