<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) <em>15</em>/19 and 21 belong to a subfamily of FGFs that function as hormones. Produced in response to specific nutritional cues, they act on overlapping sets of cell surface receptors composed of classic FGF receptors in complex with βKlotho, and regulate metabolism and related processes during periods of fluctuating energy availability. Pharmacologically, both FGF<em>15</em>/19 and FGF21 cause weight loss and improve both insulin-sensitivity and lipid parameters in rodent and primate models of metabolic disease. Recently, FGF21 was shown to have similar effects in obese patients with type 2 diabetes. We discuss here emerging concepts in FGF<em>15</em>/19 and FGF21 tissue-specific actions and critically assess their putative role as candidate targets for treating metabolic disease.

Transforming <em>growth</em> <em>factor</em> beta (TGFβ) induced differentiation of human lung <em>fibroblasts</em> to myo<em>fibroblasts</em> is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription <em>factors</em>, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands inhibit TGFβ-mediated differentiation of human lung <em>fibroblasts</em> to myo<em>fibroblasts</em> via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway leading to phosphorylation of Akt(S473). Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and <em>15</em>-deoxy-(12,14)-<em>15</em>d-prostaglandin J(2) (<em>15</em>d-PGJ(2)), inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF) <em>fibroblasts</em>, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A(1) (PGA(1)), a structural analogue of <em>15</em>d-PGJ(2) with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of <em>15</em>d-PGJ(2) and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of anti-fibrotic therapeutics.

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor EGL-<em>15</em>, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.

Tissue fibrosis results, in part, from an interaction between <em>growth</em> regulatory molecules released by mononuclear phagocytes and <em>fibroblasts</em>. In the chronic interstitial lung disorders, alveolar macrophages, the mononuclear phagocytes of the lung, are known to spontaneously release two <em>growth</em> <em>factors</em> for <em>fibroblasts</em>, fibronectin and alveolar macrophage-derived <em>growth</em> <em>factor</em> (AMDGF) that together stimulate nonreplicating lung <em>fibroblasts</em> to divide. In addition to these two primary <em>growth</em> promoting signals, alveolar macrophages are able to release other mediators that may have a potential role in modulating lung <em>fibroblast</em> replication in response to these primary signals, including interferon gamma (IFN gamma), prostaglandin E2 (PGE2), and interleukin 1 (IL-1). To evaluate this possibility, we examined the effect of each of these other mediators on lung <em>fibroblast</em> replication in response to fibronectin and AMDGF in serum-free, defined medium. IFN gamma had no effect on <em>fibroblast</em> replication. In contrast, PGE2 resulted in a dose-dependent inhibition of <em>fibroblast</em> replication in response to fibronectin and AMDGF with 50% of the maximum inhibition observed at a PGE2 concentration of less than 10 ng/ml. IL-1, while not active as a primary <em>growth</em> promoting signal, at concentrations of 4-10 U/ml, augmented <em>fibroblast</em> replication in response to fibronectin and AMDGF by 10 to <em>15</em>%. Temporally, the <em>growth</em> augmenting effect of IL-1 occurred early in the G1 phase of the cell cycle. These data indicate that lung <em>fibroblast</em> replication in response to two of the primary <em>growth</em> promoting signals spontaneously released by alveolar macrophages in the interstitial lung disorders, while uninfluenced by IFN gamma, can be inhibited by PGE2 and modestly augmented by IL-1. Understanding the relevant <em>fibroblast</em> <em>growth</em> modulatory signals within the alveolar microenvironment in the chronic interstitial disorders may lead to rational therapeutic strategies designed to interrupt the fibrotic process.

A major adaptation to exercise is new capillary formation in skeletal muscle. On the basis of angiogenesis in tumors and during development, several angiogenic <em>growth</em> <em>factors</em> may be involved, including vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1). In 9-wk-old female Wistar rats, mRNA expression for these three <em>growth</em> <em>factors</em> in gastrocnemius muscle was examined by quantitative Northern analysis after a single 1-h run at <em>15</em> or 20 m/min at 10 degrees incline in room air. A third group ran at <em>15</em> m/min in 12% O2, and resting control groups were included at inspired O2 fractions of 0.21 and 0.12. Exercise significantly increased mRNA levels two- to fourfold, which was evident over the first 4 h postexercise; by 8 and 24 h, mRNA levels returned to baseline. For all three <em>factors</em>, mRNA levels were significantly higher after exercise at 20 than at <em>15</em> m/min. Hypoxia at rest doubled VEGF and TGF-beta 1 message but had no effect on bFGF. Hypoxic exercise further raised VEGF mRNA levels but had no effect on the other <em>factors</em>. We suggest that VEGF, bFGF, and TGF-beta 1 may be involved in the angiogenic response to exercise and that reduced intracellular PO2 (as occurs during normoxic exercise) may be part of the stimulus to such <em>growth</em> <em>factor</em> production.

BACKGROUND

Vascular calcification has detrimental consequences on chronic kidney disease (CKD) patients, yet its pathogenesis is not fully understood. Fibroblast growth factor-23 (FGF-23) is involved in the regulation of mineral metabolism which may in turn affect vascular calcification. Data on the relationship between FGF-23 and peripheral vascular calcification, using conventional radiographs, are conflicting, and less is known about its relation to aortic calcification. We conducted this study to investigate the relationship between FGF-23 and aortic calcification in a standard haemodialysis setting.

METHODS

The study included 65 haemodialysis patients (46 prevalent and 19 incident) on a three times 4-h dialysis schedule as well as 15 controls. Those with diabetes, oral anticoagulation or parathyroidectomy were excluded. Intact FGF-23, parathormone, lipids, calcium and phosphorus were measured. Aortic calcification index (ACI) was assessed by a non-contrast computerized tomography (CT) of the abdominal aorta.

RESULTS

FGF-23 levels were higher among haemodialysis patients (4681.3 +/- 3906.1 pg/mL) compared to controls (98.2 +/- 51.9 pg/mL), P = 0.005. ACI was higher in haemodialysis patients (14.1 +/- 12) than controls (3.2 +/- 3.6), P = 0.009. FGF-23 (P < 0.0001) and systolic blood pressure (BP) (P < 0.0001) were independently related to ACI in stepwise multiple regression analysis of pooled analysis of haemodialysis patients, R(2) = 0.476; in subgroup analysis, the independent factors relating to ACI among prevalent dialysis patients were systolic BP (P < 0.0001), FGF-23 (P = 0.002) and age (P = 0.012), R(2)=0.48; whereas in incident patients, only FGF-23 was associated with ACI (P = 0.007), R(2) = 0.37.

CONCLUSIONS

In haemodialysis patients, FGF-23 and ACI were significantly increased, and FGF-23 was independently associated with aortic calcification.

We report that human breast cancer cells secrete a <em>growth</em> <em>factor</em> that is biologically and immunologically similar to platelet-derived <em>growth</em> <em>factor</em> (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human <em>fibroblasts</em>. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to <em>15</em>- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like <em>growth</em> <em>factor</em> by breast cancer cell lines may be important in mediating paracrine stimulation of tumor <em>growth</em>.

Epidermal <em>growth</em> <em>factor</em> (EGF) may be important in regulating the <em>growth</em> of some breast cancer cells in vivo because of its mitogenic action on some breast cancer cell lines in vitro. Epidermal <em>growth</em> <em>factor</em> receptors (EGF-R) were measured in a series of breast tumors to determine what percentage of breast tumors express EGF-R and whether EGF-R was independent of expression of estrogen receptor and progestin receptor. Specific binding of 125I-EGF to membranes from pooled homogenates of breast tumors reached equilibrium after 45 min at 25 degrees and remained constant. Scatchard analysis of 125I-EGF binding indicated a single class of receptors with an apparent Kd of 2 nM and a binding capacity of 28 fmol/mg of membrane protein, and the binding of 125I-EGF was not effectively competed for by insulin, <em>fibroblast</em> <em>growth</em> <em>factor</em>, <em>growth</em> hormone, or prolactin. Specific binding of 125I-EGF of 1 fmol or greater/mg of membrane protein and <em>15</em>% or greater specific binding was detected in 48% of 137 unselected primary and metastatic breast tumors. The frequency distribution of EGF binding values was unimodal, with a progressive decrease in the proportion of patients with high EGF binding values. The values of EGF binding ranged from 1 to 121 fmol/mg of protein, with an arithmetic mean of 8.4 fmol/mg of protein and a geometric mean of 3.2 fmol/mg of protein. Forty-two % of 24 metastatic breast tumors were positive for EGF binding, with an arithmetic mean of 6.3 fmol/mg of protein and a geometric mean of 4.1 fmol/mg of protein. The magnitude of EGF binding in individual tumors was independent of either estrogen receptor or progestin receptor levels, although the highest quantities of EGF binding were expressed by tumors lacking steroid receptors. Approximately 20% of the tumors in the study were EGF-R-positive and ER-negative, suggesting that the <em>growth</em> of these tumors may be regulated predominantly by a peptide hormone (EGF) rather than a steroid hormone (estrogen). EGF binding did not correlate significantly with age of the patients. Correlation analysis between EGF binding and the percentage of malignant and nonmalignant cell types present in sections of tumor adjacent to the area assayed for EGF binding indicated that the percentage of malignant cells is an important <em>factor</em> in determining the amount of EGF binding in tumor homogenates. The recent discovery of transforming <em>growth</em> <em>factors</em> which interact with the EGF-receptor system suggests additional roles for EGF receptors in breast cancer.

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5(-/-) mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease.

Capillary endothelial cells can be switched between <em>growth</em> and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its <em>growth</em>-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-<em>15</em> h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent <em>growth</em> control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and <em>growth</em>, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed when cytoskeletal stiffness was measured directly in living cells using magnetic twisting cytometry. These results emphasize the importance of matrix-dependent changes in cell and nuclear shape as well as higher order structural interactions between different cytoskeletal filament systems for control of capillary cell <em>growth</em> during angiogenesis.

OBJECTIVE

To investigate <em>factors</em> involved in inflammation of the prostate besides IL-<em>15</em>, we screened prostatic cells and tissues for IL-17 and IL-17 receptor expression.

METHODS

Normal prostate (n = 1), BPH (n = 19), and carcinoma (CaP, n = 12) specimens were screened for IL-17, IL-17 receptor, CD45, IL-6, and IL-8 mRNA expression. The carcinoma cell lines DU145, PC3, LNCaP, and BPH-epithelial (EC), stromal cell (SC) preparations, and BPH-T-cell lines were analyzed for IL-17 production by RT-PCR and ELISA. The effect of IL-17 on IL-6, IL-8, TGF-beta1, and fibroblast growth factor (FGF-2) mRNA expression and/or release of SC was analyzed using real-time PCR and/or ELISA. Immunohistochemistry was used to localize both IL-17 and IL-17 receptor.

RESULTS

In the normal prostate, IL-17 expression was very weak and restricted to lymphocytes. In 79% of BPH and 58% of CaP specimens, IL-17 mRNA and protein expression was increased. IL-17 mRNA expression could be shown for activated BPH-T-cells and to some extend for BPH-EC. Expression of IL-17 receptor was ubiquitous. Release of IL-17 was shown only for activated BPH-T-cells. IL-17 stimulated expression of IL-6 (13-fold) and IL-8 (26-fold) by prostatic BPH-SC. In situ, however, the amount of IL-17mRNA in BPH-tissue did not correlate with the amount of IL-6 and IL-8 mRNA. In CaP tissue, significant correlation was found only between the amount of IL-6 and IL-8 mRNA.

CONCLUSIONS

Activated BPH-T-cells abundantly express IL-17. The increase of IL-17 in BPH-tissues goes hand in hand with elevated levels of IL-<em>15</em>, a pro-inflammatory cytokine with T-cell growth factor properties. A clinical relevance of increased IL-17 expression under pathological conditions is suggested by the demonstration of significant upregulation of IL-6 and IL-8 production of prostatic SC by IL-17.

Vascular permeability <em>factor</em> (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor <em>growth</em>. Vascular permeability <em>factor</em> also promotes the <em>growth</em> of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to <em>15</em>-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human <em>fibroblasts</em>, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC <em>growth</em>-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand <em>factor</em> release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand <em>factor</em> release.

Previous studies have suggested a role for c-fos in cellular differentiation in fetal membranes, haematopoietic cells and teratocarcinoma stem cells. In other cell types, such as <em>fibroblasts</em>, c-fos expression is normally very low, but is rapidly induced by peptide <em>growth</em> <em>factors</em>, implicating c-fos in <em>growth</em> control mechanisms. Here, we show that the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced macrophage-like differentiation of HL60 human promyelocytic precursor cells is accompanied by the induction of both c-fos mRNA and protein within <em>15</em> min after treatment, suggesting a functional role for c-fos in this differentiation system. In quiescent terminally differentiated macrophages, expression of c-fos is inducible by the macrophage-specific <em>growth</em> <em>factor</em> colony-stimulating <em>factor</em>-1 (CSF-1). The kinetics of c-fos induction, however, are entirely different from those in <em>growth</em> <em>factor</em>-stimulated <em>fibroblasts</em>, supporting the view that the c-fos gene product may serve different functions in different cell types.

BACKGROUND

Hepatocellular carcinoma (HCC) is a hypervascular malignancy. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin (ANG) are important angiogenic factors of neoangiogenesis. This study investigated the predictive value of serum VEGF, bFGF, and ANG in tumor recurrence, disease-free survival (DFS), and overall survival (OS) in HCC patients.

METHODS

Preoperative serum VEGF, bFGF, and ANG were measured in 98 patients with resectable HCC and in 15 healthy controls. The median follow-up time was 43 months.

RESULTS

Preoperative serum VEGF was increased in patients with resectable HCC compared with healthy controls (P <.05). Increased serum VEGF was correlated with tumor recurrence (P =.001). Univariate analysis showed that serum VEGF, tumor-node-metastasis stage, tumor size and number, macroscopic portal vein invasion, and microscopic vascular invasion were correlated with OS and DFS. Serum bFGF and ANG were not associated with survival. Multivariate analysis showed that serum VEGF was the most significant predictor of DFS (relative risk, 2.35; 95% confidence interval, 1.26-4.39; P =.007) and OS (relative risk, 3.44; 95% confidence interval, 1.81-6.57; P <.001) in HCC patients after surgical resection.

CONCLUSIONS

Preoperative serum VEGF is a significant independent predictor of tumor recurrence, DFS, and OS in patients with resectable HCC.

1. It is now clear that members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family have multiple roles during the formation of the central nervous system (CNS). 2. There are at least 23 members of the FGF family and, of these, 10 are expressed in the developing CNS, along with four FGF receptors (FGFR-1-4). 3. The present review discusses the roles of these FGFs, with emphasis on FGF-2, FGF-8, FGF-<em>15</em> and FGF-17. <em>Fibroblast</em> <em>growth</em> <em>factors</em>-2 and -<em>15</em> are generally expressed throughout the developing CNS, whereas FGF-8 and FGF-17 are tightly localized to specific regions of the developing brain and are only expressed in the embryo during the early phases of proliferation and neurogenesis. 4. Expression studies on FGFRs in the chick and mouse indicate that FGFR-1 is most generally expressed, whereas FGFR-2 and FGFR-3 show highly localized but changing patterns of expression throughout CNS development. The FGFR-4 has been localized to the developing CNS in fish but not at a detailed level, as yet, in chick or mouse. 5. A picture is emerging from these studies that particular FGFs signal through specific receptors in a highly localized manner to regulate the development of different regions of the brain. 6. This picture has been demonstrated so far for the developing cortex (FGF-2-/- mice), the forebrain and midbrain (FGF-8 hypomorphs) and the cerebellum (FGF-17/FGF-8 mutant mice). In addition, generation of mutant animals deleted for FGFR-1 and FGFR-2b IIIb demonstrate their importance in FGF signalling. 7. However, there are significant gaps in our knowledge of the localization of members of the FGF family and their receptors. More detailed information on the spatio-temporal mapping of FGFs and FGFR isoforms is required in order to understand the molecular mechanisms through which FGFs signal.

We have shown that a type I phosphatidylinositol (PI) kinase activity is associated with the epidermal <em>growth</em> <em>factor</em> (EGF) receptor in a mouse <em>fibroblast</em> cell line expressing human EGF receptors (NRHER5) and that this activity increases dramatically upon treatment of cells with physiologically relevant concentrations of EGF. EGF stimulated a time-dependent increase in EGF receptor-associated PI kinase activity measured in EGF receptor immunoprecipitates. Activation was detected <em>15</em> min after the addition of EGF, and it peaked between 1 and 2 hr. Activation of PI kinase was detected with EGF concentrations as low as 10 pM and maximal stimulation occurred at approximately 1 nM. Analysis of deacylated PI phosphate products, and inhibition of the PI kinase activity by nonionic detergent, indicated that the PI kinase described here was type I or PI 3' kinase. These results demonstrate the regulation of a type I PI kinase by EGF and suggest a potential role in the EGF receptor signal transduction pathway.

This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, <em>15</em>, 30, and 45 (total 16 mg: 4 x 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.0<em>15</em>] and major amputations (HR 0.371; P = 0.0<em>15</em>). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.

Treatment of quiescent human embryonic lung <em>fibroblast</em>ic cells (TIG-3) with 10 nM epidermal <em>growth</em> <em>factor</em> (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at <em>15</em> min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several <em>growth</em> <em>factors</em>, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human <em>fibroblasts</em>.

The product of the c-myc proto-oncogene is an important positive regulator of cell <em>growth</em> and proliferation. Recently, c-Myc has also been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or <em>growth</em> <em>factors</em>. To further examine Myc-induced apoptosis, we coexpressed the proto-oncogene bcl2, which has been shown to block apoptosis in other systems, with c-myc in serum-deprived Rat 1a <em>fibroblasts</em>. Here we report that ectopic expression of bcl2 specifically blocks apoptosis induced by constitutive c-myc expression. Constitutive c-myc expression in serum-deprived Rat 1a cells caused a>> <em>15</em>-fold increase in the number of dead cells, accompanied by DNA fragmentation. However, coexpression of bcl2 with c-myc in these cells led to a 10-fold increase in the number of live cells and a significant decrease in DNA fragmentation. Thus, Bcl-2 effectively inhibits Myc-induced apoptosis in serum-deprived Rat 1a <em>fibroblasts</em> without blocking entry into the cell cycle. These results imply that apoptosis serves as a protective mechanism to prevent tumorigenicity elicited by deregulated Myc expression. This protective mechanism is abrogated, however, by Bcl-2 and therefore may explain the synergism between Myc and Bcl-2 observed in certain tumor cells.

OBJECTIVE

The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer.

METHODS

We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity.

RESULTS

TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K-AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo.

CONCLUSIONS

Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers.

OBJECTIVE

The involvement of cytokines and chemokines in vitreous fluid is important in the development and progression of diabetic retinopathy (DR) and central retinal vein occlusion (CRVO). In this study, the concentrations of cytokines and chemokines in the vitreous fluid of eyes with DR and CRVO were measured and compared.

METHODS

We studied 76 eyes with proliferative DR and diabetic macular edema (DR group), 10 eyes with CRVO (CRVO group), and 23 eyes with an epiretinal membrane and macular hole (control group), among a series of 160 eyes from which vitreous fluid samples were collected during vitrectomy. The vitreous fluid samples were collected by suction with a vitreous cutter at the initial stage of vitrectomy. Twenty-seven different cytokines and chemokines were measured simultaneously using an array system (Bio-Plex(®)) with beads combined with antibodies (Bio-Rad), as follows: interleukin (IL)-1β, IL-1 receptor agonist, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-<em>15</em>, IL-17, eotaxin, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, granulocyte colony-stimulating <em>factor</em> (G-CSF), granulocyte/macrophage colony-stimulating <em>factor</em> (GM-CSF), interferon (IFN)-γ, interferon-inducible 10-kDa protein (IP-10), monocytochemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB, regulated upon activation, normal T cell expressed and secreted, tumor necrosis <em>factor</em> alpha (TNF-α), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF).

RESULTS

Compared to the control group, the levels of IL-6, IL-8, IL-10, IL-13, IP-10, MCP-1, MIP-1β, PDGF and VEGF in the vitreous fluid were significantly higher in the DR group, while the levels of IL-1β, IL-2, IL-5, IL-8, IL-9, IL-10, IL-12, IL-13, eotaxin, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1β, TNF-α and VEGF were significantly higher in the CRVO group. Compared to the DR group, IL-2, IL-9, IL-12, MCP-1 and IFN-γ were significantly elevated in the CRVO group. Multivariate regression analysis revealed that among 6 factors correlated to VEGF in the DR group, IL-10 and IL-13 were more positively correlated and PDGF was most inversely correlated to VEGF.

CONCLUSIONS

In addition to inflammatory cytokines and neurotrophic factors such as VEGF, anti-inflammatory cytokines such as IL-10 and IL-13 may be involved more in the pathogenesis of DR and CRVO than in other diseases; cytokines and chemokines may also be correlated to VEGF in the vitreous fluid. It is also suggested that the inflammatory reaction may be more activate in CRVO than in DR.

Changes in human endometrium are essential to allow the establishment of pregnancy. These changes are induced in vivo by progesterone, and include appearance within the tissue of a specific uterine natural killer cell, characterized by an abundant expression of CD56. Changes also occur in the stromal cells, which undergo a characteristic decidualization reaction. Decidualized stromal cells are derived from the <em>fibroblast</em>-like cells within the endometrium, which maintain their progesterone receptors in the presence of progesterone. Prolonged exposure to progesterone induces a rounded cell characterized by release of prolactin and insulin-like <em>growth</em> <em>factor</em> binding protein-1 (IGFBP-1), and expression of tissue <em>factor</em>. Additional changes include the secretion of interleukin (IL)-<em>15</em>, vascular endothelial <em>growth</em> <em>factor</em>, and surface expression of zinc dependent metalloproteinases such as CD10 and CD13. In vitro, elevated intracellular cAMP as well as progesterone is necessary for decidualization. In vivo, these conditions may be provided by progesterone from the corpus luteum, by prostaglandin E, a stimulator of adenyl cyclase, and relaxin, which has recently been shown to be a phosphodiesterase inhibitor. Given the co-distribution of uterine natural killer cells and decidualized stromal cells, a mutual interaction might provide the correct regulatory environment for successful implantation, and penetration of the maternal blood vessels by trophoblastic cells.

OBJECTIVE

Roux-en-Y gastric bypass (RYGB) in humans can remit type 2 diabetes, but the operative mechanism is not completely understood. In mice, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>15</em> (FGF19 in humans) regulates hepatic bile acid (BA) production and can also resolve diabetes. In this study, we tested the hypothesis that the FGF19-BA pathway plays a role in the remission of human diabetes after RYGB surgery.

METHODS

Cohorts of diabetic and nondiabetic individuals of various body weights were used. In addition, RYGB patients without diabetes (No-Diabetes), RYGB patients with diabetes who experienced remission for at least 12 months after surgery (Diabetes-R), and RYGB patients with diabetes who did not go into remission after surgery (Diabetes-NoR) were studied. Circulating FGF19 and BA levels, hepatic glycogen content, and expression levels of genes regulating the FGF19-BA pathway were compared among these groups of patients using pre- and postoperative serum samples and intraoperative liver biopsies.

RESULTS

Preoperatively, patients with diabetes had lower FGF19 and higher BA levels than nondiabetic patients, irrespective of body weight. In diabetic patients undergoing RYGB, lower FGF19 levels were significantly correlated with increased hepatic expression of the cholesterol 7alpha-hydroxylase 1 (CYP7A1) gene, which modulates BA production. Following RYGB surgery, however, FGF19 and BA levels (particularly cholic and deoxycholic acids) exhibited larger increases in Diabetic-R patients compared with nondiabetic and Diabetic-NoR patients.

CONCLUSIONS

Taken together, the baseline and postoperative data implicate the FGF19-CYP7A1-BA pathway in the etiology and remission of type 2 diabetes following RYGB surgery.

During the development of the egg-laying system in Caenorhabditis elegans hermaphrodites, central gonadal cells organize the alignment of the vulva with the sex myoblasts, the progenitors of the egg-laying muscles. A <em>fibroblast</em> <em>growth</em> <em>factor</em> [EGL-17(FGF)] and an FGF receptor [EGL-<em>15</em>(FGFR)] are involved in the gonadal signals that guide the migrations of the sex myoblasts. Here we show that EGL-17(FGF) can act as an instructive guidance cue to direct the sex myoblasts to their final destinations. We find that egl-17 reporter constructs are expressed in the primary vulval cell and that EGL-17(FGF) expression in this cell correlates with the precise positioning of the sex myoblasts. We postulate that EGL-17(FGF) helps to coordinate the development of a functional egg-laying system, linking vulval induction with proper sex myoblast migration.