Numerous studies of the proliferative effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in culture, including neonatal and adult hippocampal precursors, suggest that the <em>factor</em> plays a ubiquitous and life-long role in neurogenesis. In contrast, in vivo, bFGF is devoid of effects on neurons in mature hippocampus, raising the possibility that bFGF exhibits developmental stage-specific activity in the complex animal environment. To define neurogenetic effects in the newborn, a single subcutaneous injection of bFGF (20 ng/gm) was administered to postnatal day 1 (P1) rats, and hippocampal DNA content was quantified: bFGF elicited an increase in total DNA throughout adulthood, by 48% at P4, 25% at P22, and 17% at P180, suggesting that bFGF increases hippocampal cell number. To define mechanisms, bromodeoxyuridine (BrdU) was injected at P1 and mitotically labelled cells were assessed at P22: there was a twofold increase in BrdU-positive cells in the dentate granule cell layer (GCL), indicating that bFGF enhanced the generation of neurons, or neuronogenesis, from a cohort of precursors. Moreover, enhanced mitosis and survival led to a 33% increase in absolute GCL neuron number, suggesting that neuron production depends on environmental levels of bFGF. To evaluate this possibility, bFGF-knockout mice were analyzed: hippocampal DNA content was decreased at all ages examined (P3, -42%; P21, -28%; P360, -18%), and total GCL neuron and glial fibrillary acidic protein (GFAP)-positive cell number were decreased by 30%, indicating that bFGF is necessary for normal hippocampal neurogenesis. We conclude that environmental levels of bFGF regulate neonatal hippocampal neurogenesis. As adult hippocampal neuronogenesis was unresponsive to bFGF manipulation in our previous study [Wagner, J.P., Black, I.B. & DiCicco-Bloom, E. (<em>19</em>99) J. Neurosci., <em>19</em>, 6006], these observations suggest distinct, stage-specific roles of bFGF in the dentate gyrus granule cell lineage.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-<em>19</em>-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal <em>factor</em> in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.

<em>Fibroblast</em>/myo<em>fibroblast</em> expansion is critical in the pathogenesis of pulmonary fibrosis. To date, most research has focused on profibrotic mediators, whereas studies on antifibrotic <em>factors</em> are scanty. In this study, we explored the effects of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1) and FGF-1 plus heparin (FGF-1+H) on <em>fibroblast</em> <em>growth</em> rate, apoptosis, and myo<em>fibroblast</em> differentiation. Heparin was used because it participates in FGF-1 signaling. <em>Growth</em> rate was evaluated by WST-1 colorimetric assay, DNA synthesis by [(3)H]thymidine incorporation, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase 3. Expression of alpha-smooth muscle actin (alpha-SMA) was examined by immunocytochemistry, flow cytometry, real-time PCR, and immunoblotting. Despite the induction of DNA synthesis, FGF-1+H significantly reduced <em>fibroblast</em> <em>growth</em> rate. This correlated with a significant increase in apoptosis, evaluated by TUNEL (41.6 +/- 1.4% vs. 12.5 +/- 0.6% from controls; P < 0.01) and cleaved caspase 3 (295 +/- 32 vs. 200 +/- <em>19</em> ng/10(6) cells from controls; P < 0.05). Double immunostaining (alpha-SMA-TUNEL) revealed that the levels of induced apoptosis were similar in <em>fibroblasts</em> and myo<em>fibroblasts</em>. FGF-1+H inhibited the effect of TGF-beta1 on myo<em>fibroblast</em> differentiation. alpha-SMA-positive cells were reduced by immunocytochemistry from 44.5 +/- 6.5% to 10.9 +/- 1.9% and by flow cytometry from 30.6 +/- 2.5% to 7.7 +/- 0.6% (P < 0.01). Also, FGF-1+H significantly inhibited the TGF-beta1 induction of alpha-SMA quantified by real-time PCR and Western blot. This decrease was associated with a 35% reduction in TGF-beta1-induced collagen gel contraction. The effect of FGF-1+H was mediated by a significant decrease of TGF-beta1-induced Smad2 phosphorylation. FGF-1 alone exhibited similar but lower effects. These findings suggest that FGF-1 can have an antifibrogenic role, inducing apoptosis of <em>fibroblasts</em> and inhibiting myo<em>fibroblast</em> differentiation.

This chapter will review the various biological actions of the mouse <em>fibroblast</em> <em>growth</em> <em>factor</em> 15 (Fgf15) and human <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>). Unlike other members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, the Fgf15 and FGF<em>19</em> orthologs do not share a high degree of sequence identity. Fgf15 and FGF<em>19</em> are members of an atypical subfamily of FGFs that function as hormones. Due to subtle changes in tertiary structure, these FGFs have low heparin binding affinity enabling them to diffuse away from their site of secretion and signal to distant cells. FGF signaling through the FGF receptors is also different for this sub-family, requiring klotho protein co<em>factor</em>s rather than heparin sulfate proteoglycan. Mouse Fgf15 and human FGF<em>19</em> play key roles in enterohepatic signaling, regulation of liver bile acid biosynthesis, gallbladder motility and metabolic homeostasis.

OBJECTIVE

Reports of increased circulating fibroblast growth factor 21 (FGF21) levels in obesity indicate that FGF21 may be implicated in body weight homeostasis. We sought to investigate the existence of FGF21 in human cerebrospinal fluid (CSF) and, if present, the relationship between CSF FGF21 with body adiposity and metabolic parameters.

METHODS

CSF and corresponding plasma FGF21 were measured by an enzyme-linked immunosorbent assay (18 men and 20 women, aged 19-80 years, and BMI 16.2-38.1 kg/m(2)) and correlated to body adiposity and metabolic parameters.

RESULTS

CSF and plasma FGF21 increased in particular with rising BMI and fat mass. In CSF, FGF21 was detectable at concentrations ~40% that of plasma levels. CSF and plasma FGF21 levels were significantly positively correlated with BMI and fat mass, body weight, plasma insulin, and homeostasis model assessment of insulin resistance. Plasma FGF21 levels were significantly negatively correlated with plasma adiponectin. When subjected to multiple regression analysis, only fat mass was predictive of plasma FGF21 (β = 0.758; P = 0.004) and CSF FGF21 (β = 0.767; P = 0.007). The CSF-to-plasma FGF21 ratio was significantly negatively correlated with BMI, fat mass, and plasma FGF21. Subjects in the highest plasma FGF21 quintile had a lower CSF-to-plasma FGF21 ratio (12.7% [9.7-14.9%]) compared with those in the lowest plasma FGF21 quintile (94.7% [37.3-99.8%]) (P < 0.01).

CONCLUSIONS

Our observations have important implications with respect to the potential central actions of FGF21. Future research should seek to clarify whether FGF21 would be beneficial in the management of obesity and its metabolic complications.

Myocardial ischemia (MI) activates innate cardioprotective mechanisms, enhancing cardiomyocyte tolerance to ischemia. Here, we report a MI-activated liver-dependent mechanism for myocardial protection. In response to MI in the mouse, hepatocytes exhibited 6- to <em>19</em>-fold upregulation of genes encoding secretory proteins, including α-1-acid glycoprotein (AGP)2, bone morphogenetic protein-binding endothelial regulator (BMPER), chemokine (C-X-C motif) ligand 13, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)21, neuregulin (NRG)4, proteoglycan 4, and trefoil <em>factor</em> (TFF)3. Five of these proteins, including AGP2, BMPER, FGF21, NRG4, and TFF3, were identified as cardioprotective proteins since administration of each protein significantly reduced the fraction of myocardial infarcts (37 ± 9%, 34 ± 7%, 32 ± 8%, 39 ± 6%, and 31 ± 7%, respectively, vs. 48 ± 7% for PBS at 24 h post-MI). The serum level of the five proteins elevated significantly in association with protein upregulation in hepatocytes post-MI. Suppression of a cardioprotective protein by small interfering (si)RNA-mediated gene silencing resulted in a significant increase in the fraction of myocardial infarcts, and suppression of all five cardioprotective proteins with siRNAs further intensified myocardial infarction. While administration of a single cardioprotective protein mitigated myocardial infarction, administration of all five proteins furthered the beneficial effect, reducing myocardial infarct fractions from PBS control values from 46 ± 6% (5 days), 41 ± 5% (10 days), and 34 ± 4% (30 days) to 35 ± 5%, 28 ± 5%, and 24 ± 4%, respectively. These observations suggest that the liver contributes to cardioprotection in MI by upregulating and releasing protective secretory proteins. These proteins may be used for the development of cardioprotective agents.

Clinical and histopathological features do not reliably distinguish between benign and malignant pheochromocytomas. Additional markers that might be useful prognostic indicators in the pathological assessment of these tumors are sought. Immunohistochemical expression of MIB-1, Bcl-2, cathepsin B, cathepsin D, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), c-met, and type IV collagenase were studied on formalin-fixed tissue from 33 nonconsecutive cases of pheochromocytoma, selected on the basis of reliable long-term follow-up, to determine associations with malignancy. The study group included 33 patients, <em>19</em> men and 14 women, with a mean age of 45 years, including five cases of neurofibromatosis (NF), three familial, and one MEN IIb. Mean follow-up was 63.2 months. Ten patients were determined to have malignant pheochromocytomas by the presence of metastatic disease. Features found to be associated with malignancy included MIB-1 labeling index (5% vs 1%) (P = .0009), male gender (90% vs 43%) (P = .008), extra-adrenal location (40% vs 9%) (P = .03), tumor weight (481 g vs 124 g) (P = .05), and young age (38 years vs 49 years) (P = .05). None of the five cases with NF were malignant (P = .04). S-100 positivity showed a significant (P = .02) but nonlinear association with benign tumors. Absent S-100 correlated with greater tumor weight. Malignancy was not associated with right versus left side or bilaterality, although bilateral tumors were smaller. C-met, bFGF, cathepsin B, cathepsin D, and collagenase were strongly expressed in most tumors and were not predictive of outcome, nor was bcl-2, which was variably expressed. Using multiple logistic regression with malignancy as the dependent variable, MIB-1 continued to show a significant association with malignancy (P = .005) independent of any association with sex, age, or extra-adrenal location. Using a cutoff value of MIB-1 labeling of greater than 3% yielded a specificity of 100% and a sensitivity of 50% in predicting malignancy.

Cholesterol 7alpha-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3'-untranslated region (3'-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em>. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3'-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3'-UTR reporter plasmids, and mutations of miRNA binding sites in 3'-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF<em>19</em> inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

BACKGROUND

Circulating angiogenic factors in patients with colorectal cancer liver metastases may promote tumor growth and contribute to liver regeneration after partial hepatectomy.

METHODS

We analyzed blood samples from 26 patients with colorectal cancer liver metastases before and after liver resection and used samples from 20 healthy controls as a reference. Plasma levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) were measured, and levels were correlated with recurrence.

RESULTS

The median preoperative levels of all four factors were significantly higher and more variable in colorectal cancer liver metastasis patients than in controls. HGF and bFGF levels increased significantly 3 days and 1 month after hepatectomy, respectively, and returned to near preoperative levels at 3 months. Postoperative VEGF and EGF levels remained relatively stably increased over 3 months. After a median follow-up of 19 months, 10 patients (42%) experienced recurrence. Higher preoperative VEGF and HGF levels correlated with subsequent recurrence (P = .018 and .021, respectively), and a preoperative adjusted total value of all four factors accurately identified patients at low, moderate, and high risk of recurrence (P = .034). Patients who experienced disease recurrence also had relatively higher bFGF levels 3 months after operation (P = .035).

CONCLUSIONS

Plasma angiogenic factors are increased in patients with colorectal cancer liver metastases and remain increased at least 3 months after partial hepatectomy. Measurement of certain factors before and after hepatic resection can predict recurrence. Targeted biological agents may counteract the tumor-promoting effects of these circulating factors on subclinical disease.

The heart provides the body with oxygen and nutrients and assists in the removal of metabolic waste through the blood vessels of the circulatory system. It is the first organ to form during embryonic morphogenesis. FGFs with diverse functions in development, health, and disease are signaling proteins, mostly as paracrine <em>growth</em> <em>factors</em> or endocrine hormones. The human/mouse FGF family comprises 22 members. Findings obtained from mouse models and human diseases with FGF signaling disorders have indicated that several FGFs are involved in heart development, health, and disease. Paracrine FGFs including FGF8, FGF9, FGF10, and FGF16 act as paracrine signals in embryonic heart development. In addition, paracrine FGFs including FGF2, FGF9, FGF10, and FGF16 play roles as paracrine signals in postnatal heart pathophysiology. Although FGF15/<em>19</em>, FGF21, and FGF23 are typical endocrine FGFs, they mainly function as paracrine signals in heart development or pathophysiology. In heart diseases, serum FGF15/<em>19</em> levels or FGF21 and FGF23 levels decrease or increase, respectively, indicating their possible roles in heart pathophysiology. FGF2 and FGF10 also stimulate the cardiac differentiation of cultured stem cells and cardiac reprogramming of cultured <em>fibroblasts</em>. These findings provide new insights into the roles of FGF signaling in the heart and potential therapeutic strategies for cardiac disorders.

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and null (n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null (P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 μl (P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 μl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to <em>19</em> ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). <em>Fibroblasts</em> isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue <em>growth</em> <em>factor</em> (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in <em>fibroblast</em> gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired <em>fibroblast</em> activation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em> and FGF21 are hormones that regulate metabolic processes particularly during feeding or starvation, thus ultimately influencing energy production. FGF<em>19</em> is secreted by the intestines during feeding and negatively regulates bile acid synthesis and secretion, whereas FGF21 is produced in the liver during fasting and plays a crucial role in regulating glucose and lipid metabolism, as well as maintaining energy homeostasis. FGF<em>19</em> and FGF21 are regarded as late-acting hormones because their functions are only used after insulin and glucagon have completed their actions. Although FGF<em>19</em> and FGF21 are activated under different conditions, they show extensively functional overlap in terms of improving glucose tolerance, insulin sensitivity, weight loss, and lipid, and energy metabolism, particularly in pathological conditions such as diabetes, obesity, metabolic syndrome, and cardiovascular and renal diseases. Most patients with these metabolic diseases exhibit reduced serum FGF<em>19</em> levels, which might contribute to its etiology. In addition, the simultaneous increase in serum FGF21 levels is likely a compensatory response to reduced FGF<em>19</em> levels, and the 2 proteins concertedly maintain metabolic homeostasis. Here, we review the physiological and pharmacological cross talk between FGF<em>19</em> and FGF21 in relation to the regulation of endocrine metabolism and various chronic diseases.

<AbstractText>Combined administration of anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal <em>growth</em> <em>factor</em> receptor 2 (HER2; ErbB2), is an effective treatment for HER2-positive breast cancer. However, both agents are associated with cardiac toxicity. Human cardiac-resident mesenchymal progenitor cells (CPCs) secrete extracellular vesicles including nanosized exosomes which protect against myocardial ischemia. Here, we investigated the effects of these exosomes using a novel model of Dox/Trz-mediated cardiotoxicity.</AbstractText><AbstractText>CPCs were derived from cardiac atrial appendage specimens from patients who underwent heart surgery for heart valve disease and/or ischemic heart disease, and exosomes were purified from CPC conditioned media. Proteomics analyses revealed that CPC exosomes contained multiple proteins involved in redox processes. Dox/Trz induced a significant increase in reactive oxygen species (ROS) in rat cardiomyocytes, which was prevented by CPC exosomes. In vivo, rats received 6 doses of Dox (days 1-11), followed by 6 doses of Trz (days <em>19</em>-28). Three doses of either exosomes or exosome suspension vehicle were injected intravenously on days 5, 11 and <em>19</em>. Dox/Trz induced myocardial fibrosis, CD68+ inflammatory cell infiltrates, inducible nitric oxide synthase (iNOS) expression, and left ventricular (LV) dysfunction. CPC exosomes prevented these effects. These vesicles were highly enriched in miR-146a-5p compared with human dermal <em>fibroblast</em> exosomes. Dox upregulatedTraf6 and Mpo, two known miR-146a-5p target genes (which encode signaling mediators of inflammatory and cell death axes) in myocytes. CPC exosomes suppressed miR-146a-5p target genesTraf6, Smad4, Irak1, Nox4 and Mpo in Dox-treated cells. Specific silencing of miR-146a-5p abrogated exosome-mediated suppression of those genes leading to an increase in Dox-induced cell death.</AbstractText><AbstractText>Human CPC exosomes attenuate Dox/Trz-induced oxidative stress in cardiomyocytes. Systemic administration of these vesicles prevents Dox/Trz cardiotoxicity in vivo. miR-146a-5p mediates some of the benefits of exosomes in this setting.</AbstractText>

BACKGROUND

Intestinal bacteria are known to regulate bile acid (BA) homeostasis via intestinal biotransformation of BAs and stimulation of the expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> through intestinal nuclear farnesoid X receptor (FXR). On the other hand, BAs directly regulate the gut microbiota with their strong antimicrobial activities. It remains unclear, however, how mammalian BAs cross-talk with gut microbiome and shape microbial composition in a dynamic and interactive way.

RESULTS

We quantitatively profiled small molecule metabolites derived from host-microbial co-metabolism in mice, demonstrating that BAs were the most significant factor correlated with microbial alterations among all types of endogenous metabolites. A high-fat diet (HFD) intervention resulted in a rapid and significant increase in the intestinal BA pool within 12 h, followed by an alteration in microbial composition at 24 h, providing supporting evidence that BAs are major dietary factors regulating gut microbiota. Feeding mice with BAs along with a normal diet induced an obese phenotype and obesity-associated gut microbial composition, similar to HFD-fed mice. Inhibition of hepatic BA biosynthesis under HFD conditions attenuated the HFD-induced gut microbiome alterations. Both inhibition of BAs and direct suppression of microbiota improved obese phenotypes.

CONCLUSIONS

Our study highlights a liver-BA-gut microbiome metabolic axis that drives significant modifications of BA and microbiota compositions capable of triggering metabolic disorders, suggesting new therapeutic strategies targeting BA metabolism for metabolic diseases.

The aim of our study was to measure serum concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF-<em>19</em>) in patients with obesity (OB), obesity and type 2 diabetes mellitus (T2DM) and healthy subjects (C) at baseline and after selected interventions. We measured serum FGF-<em>19</em> levels and other biochemical and hormonal parameters in 29 OB and <em>19</em> T2DM females and 30 sex- and age-matched control subjects. The interventions were acute hyperinsulinemia during isoglycemic-hyperinsulinemic clamp (n=11 for T2DM and 10 for C), very-low calorie diet (VLCD, n=12 for OB) and 3 months treatment with PPAR-alpha agonist fenofibrate (n=11 for T2DM). Baseline serum FGF-<em>19</em> levels were significantly lower in OB relative to C group (132.1+/-12.7 vs. 202.2+/-16.7 pg/ml, p<0.05), while no significant difference was observed between T2DM and OB or control group. Acute hyperinsulinemia tended to decrease FGF-<em>19</em> levels in both healthy and T2DM subjects. Three weeks of VLCD in OB group had no significant effect on FGF-<em>19</em>, whereas three months of fenofibrate treatment markedly reduced FGF-<em>19</em> levels in T2DM patients (<em>19</em>4.58+/-26.2 vs. 107.47+/-25.0 pg/ml, p<0.05). We conclude that FGF-<em>19</em> levels in our study were at least partially dependent upon nutritional status, but were not related to parameters of glucose metabolism or insulin sensitivity.

We identified a mutation in the Diet1 gene in a mouse strain that is resistant to hyperlipidemia and atherosclerosis. Diet1 encodes a 236 kD protein consisting of tandem low-density lipoprotein receptor and MAM (meprin-A5-protein tyrosine phosphatase mu) domains and is expressed in the enterocytes of the small intestine. Diet1-deficient mice exhibited an elevated bile acid pool size and impaired feedback regulation of hepatic Cyp7a1, which encodes the rate-limiting enzyme in bile acid synthesis. In mouse intestine and in cultured human intestinal cells, Diet1 expression levels influenced the production of <em>fibroblast</em> <em>growth</em> <em>factor</em> 15/<em>19</em> (FGF15/<em>19</em>), a hormone that signals from the intestine to liver to regulate Cyp7a1. Transgenic expression of Diet1, or adenoviral-mediated Fgf15 expression, restored normal Cyp7a1 regulation in Diet-1-deficient mice. Diet1 and FGF<em>19</em> proteins exhibited overlapping subcellular localization in cultured intestinal cells. These results establish Diet1 as a control point in enterohepatic bile acid signaling and lipid homeostasis.

Bile acid diarrhea (BAD) is usually seen in patients with ileal Crohn's disease or ileal resection. However, 25% to 50% of patients with functional diarrhea or diarrhea-predominant irritable bowel syndrome (IBS-D) also have evidence of BAD. It is estimated that 1% of the population may have BAD. The causes of BAD include a deficiency in <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF-<em>19</em>), a hormone produced in enterocytes that regulates hepatic bile acid (BA) synthesis. Other potential causes include genetic variations that affect the proteins involved in BA enterohepatic circulation and synthesis or in the TGR5 receptor that mediates the actions of BA in colonic secretion and motility. BAs enhance mucosal permeability, induce water and electrolyte secretion, and accelerate colonic transit partly by stimulating propulsive high-amplitude colonic contractions. There is an increased proportion of primary BAs in the stool of patients with IBS-D, and some changes in the fecal microbiome have been described. There are several methods of diagnosing BAD, such as (75)selenium homotaurocholic acid test retention, serum C4, FGF-<em>19</em>, and fecal BA measurement; presently, therapeutic trials with BA sequestrants are most commonly used for diagnosis. Management involves the use of BA sequestrants including cholestyramine, colestipol, and colesevelam. FXR agonists such as obeticholic acid constitute a promising new approach to treating BAD.

Human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1) is a member of the beta-trefoil hyperfamily and exhibits a characteristic threefold symmetry of the tertiary structure. However, evidence of this symmetry is not readily apparent at the level of the primary sequence. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary sequence during evolution. Using intra-chain and homologue sequence comparisons for <em>19</em> members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to threefold symmetry at the level of the primary sequence. The consequences of these mutations regarding structure and stability were evaluated using a combination of X-ray crystallography and differential scanning calorimetry. The mutational effects on structure and stability can be rationalized through the characterization of "microcavities" within the core detected using a 1.0A probe radius. The results show that the symmetric constraint within the primary sequence is compatible with a well-packed core and near wild-type stability. However, despite the general maintenance of overall thermal stability, a noticeable increase in non-two-state denaturation follows the increase in primary sequence symmetry. Therefore, properties of folding, rather than stability, may contribute to the selective pressure for asymmetric primary core sequences within symmetric protein architectures.

<em>Factor</em> H (FH) of the complement system acts as a regulatory co<em>factor</em> for the <em>factor</em> I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>-heparin complex. A tenth substitution and another conserved basic residue in SCR-<em>19</em> are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney.

Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in <em>fibroblasts</em> by a variety of <em>growth</em> <em>factors</em>. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and autoimmunity, all apparently mediated by an excess of tumor necrosis <em>factor</em> alpha. We recently showed that full serum inducibility of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited serum-induced TTP expression. We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the splice sites, each resulted in the maintenance of approximately 45 and <em>19</em>%, respectively, of full serum-induced expression. In addition, deletion of intron sequences base pairs 601-655 decreased the serum-induced expression of TTP by 65%. Sequence base pairs 618-626 bound specifically to the transcription <em>factor</em> Sp1; mutation of this binding motif decreased TTP expression by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its natural location also contributes significantly to the regulated expression of this gene.

BACKGROUND

Preclinical results indicate acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) present in solid tumors as a cause of broad-spectrum chemoresistance, whereas earlier clinical studies suggest that bFGF expression is associated with opposing outcomes in patients. We investigated the relationship between FGF expression and paclitaxel activity in tumors from bladder, breast, head and neck, ovarian, and prostate cancer patients.

METHODS

Tumors (n = 96) were maintained in three-dimensional histocultures, retaining tumor-stromal interaction. Bladder tumors were treated with paclitaxel for 2 h, and the other tumors for 24 h. Antiproliferative and proapoptotic effects of paclitaxel were quantified and correlated with expression of aFGF, bFGF, P-glycoprotein (Pgp), p53, and bcl-2.

RESULTS

Fifty-one percent (49/96) and 63% (61/96) of tumors showed aFGF and bFGF staining, respectively. aFGF expression was positively correlated with tumor stage (p < 0.01), and bFGF expression with tumor grade and Pgp expression (p < 0.05). Paclitaxel inhibited antiproliferation in 86% of tumors (83/96), with an average inhibition of 46 +/- 19% (mean +/- SD) in the responding tumors. Paclitaxel also induced apoptosis in 96% of tumors (92/96), with an average apoptotic index of 12 +/- 7% in the responding tumors. aFGF expression did not correlate with tumor sensitivity to paclitaxel, whereas bFGF expression showed an inverse correlation (p < 0.01). bFGF expression was a stronger predictor of paclitaxel resistance compared to Pgp, p53, or Bcl-2.

CONCLUSIONS

These results support a role of bFGF in paclitaxel resistance in human patient tumors.

BACKGROUND

Despite a greater understanding of the molecular heterogeneity of breast cancer, current therapeutic strategies still cannot overcome the relatively poor prognosis of triple-negative breast cancer (TNBC). Deregulation of fibroblast growth factor (FGF) signaling has been found in breast cancer, and blocking this pathway has been suggested as a potential therapeutic target. We therefore evaluated the expression and copy number changes of FGF family members in TNBC.

METHODS

We retrospectively evaluated 148 primary TNBC in 2009 for FGFR1, FGFR2, and FGF2 expression by immunohistochemistry. FGFR1 and FGFR2 gene copy numbers were analyzed by fluorescence in situ hybridization. The Cancer Genome Atlas (TCGA) data was used to study correlations between gene expression and amplification or methylation of FGFR1, FGFR2, and FGF2 in basal-like TNBC.

RESULTS

FGFR1, FGFR2, and FGF2 expression were found in 16.2 % (24 of 148), 12.8 % (19 of 148), and 12.8 % (19 of 148) of TNBCs, respectively. FGFR1 gene amplification was observed in 4.1 % (6 of 145), and FGFR1 high polysomy was detected in 6.9 % (10 of 145) of the cases examined. FGFR2 gene amplification and high polysomy were identified in 4.7 % (6 of 129 cases) and 0.8 % (1 of 129 cases), respectively. FGF2 expression was found to be associated with basal-like TNBC. The expression of FGF family members and FGFR1 or FGFR2 gene amplification did not affect patient survival. TCGA data revealed that promoter methylation of the 3 genes was significantly associated with mRNA expression.

CONCLUSIONS

Even though the implications for patient outcomes are not significant, subsets of TNBCs harbor FGFR1 or FGFR2 gene amplification and FGFR1, FGFR2, or FGF2 protein overexpression.

Retention of inflammatory mediators and cells in the middle ear cleft during chronic otitis media with effusion (COME), results in ongoing inflammation with the potential for pathologic changes and hearing loss. Cytokines are glycoproteins produced by macrophages and other cells. Activities of cytokines include fever production, osteoclast, <em>fibroblast</em>, phagocyte and cytotoxic cell activation, regulation of antibody formation, and inhibition of cartilage, bone and endothelial cell <em>growth</em>. Using enzyme-linked immunospecific assays we measured levels of six cytokines in middle ear effusions (MEE) from children with COME. Significant levels of four cytokines: interleukin-1-beta (greater than 50 pg/ml), interleukin-2 (greater than 300 pg/ml), tumor necrosis <em>factor</em>-alpha (greater than 40 pg/ml), and gamma-interferon (greater than 6.25 pg/ml) were found in 51%, 54%, 63%, and <em>19</em>% of MEE, respectively. In contrast, levels of a fifth cytokine, granulocyte-macrophage colony-stimulating <em>factor</em>, and a sixth cytokine, interleukin-4, were undetectable. Age was observed to have a significant effect on the levels of specific cytokines. Interleukin-1 (IL-1) correlated inversely (P less than .02) with age such that the younger the child, the higher the level of IL-1 in MEE. Tumor necrosis <em>factor</em>-alpha (TNF) correlated directly (P less than .005) with age such that the older the child, the higher the level of TNF in MEE. Children undergoing tympanostomy on multiple occasions had average MEE TNF levels (234.2 +/- 109.1 pg/mg total protein) that were nearly 14 times higher (P less than .005) than those from children undergoing their first tympanostomy (16.9 +/- 3.0 pg/mg total protein). Thus IL-1 correlated with the early stages of COME, while TNF correlated with persistence of disease. The presence of these cytokines in MEE may be responsible for the mucosal damage, bone erosion, fibrosis, and resulting hearing loss seen in some cases of COME.

In addition to its central role in hemostasis, thrombin may play a role in inflammation and remodeling. To investigate the contribution of thrombin to allergic airway inflammation in asthma, we used an enzymatic assay to determine thrombin activity in bronchoalveolar lavage fluid obtained from <em>19</em> subjects with atopic asthma before (Day 0) and 48 hours after (Day 2) segmental bronchoprovocation with antigen. Thrombin activity increased from 0 (0, 2.9) on Day 1 to 41.1 (0.3, 75.6) U x 10(-3)/ml on Day 2 (p = 0.002) and correlated with total protein levels in lavage fluid on Day 2 (r = 0.885, p < 0.001). After antigen challenge, thrombin activity also showed significant correlations with interleukin-5 (r = 0.66, p = 0.002), transforming <em>growth</em> <em>factor</em> beta1 (r = 0.70, p < 0.001), fibronectin (r = 0.85, p < 0.001) and tissue <em>factor</em> (r = 0.55, p = 0.03) levels in lavage fluid. Furthermore, Day 2, but not Day 0 lavage fluid, induced proliferation of human airway <em>fibroblasts</em>. This mitogenic effect was significantly reduced with hirudin, a specific thrombin inhibitor. Taken together, our findings suggest that allergen-driven airway inflammation in asthma is associated with enhanced potential for <em>fibroblast</em> proliferation that is related, at least in part, to increased thrombin activity. We propose that enhanced thrombin activity provides a potential link between allergic inflammation and initiation of airway remodeling.