Pulmonary fibrosis occurs in up to 70% of scleroderma patients and progresses to cause severe restrictive lung disease in about <em>15</em>% of patients. The mechanisms that cause pulmonary fibrosis in scleroderma remain incompletely understood. Increased amounts of mRNA or protein for multiple profibrotic cytokines and chemokines have been identified in lung tissue or broncholveolar lavage samples from scleroderma patients, when compared to healthy controls. These cytokines include transforming <em>growth</em> <em>factor</em> (TGF)-beta, connective tissue <em>growth</em> <em>factor</em> (CTGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), oncostatin M (OSM), monocyte chemotactic <em>factor</em>-1 and pulmonary and activation-regulated chemokine (PARC). Potential cellular sources of these profibrotic cytokines and chemokines in scleroderma lung disease include alternatively activated macrophages, activated CD8+ T cells, eosinophils, mast cells, epithelial cells and <em>fibroblasts</em> themselves. This review summarizes the literature on involvement of cytokines and chemokines in the development of pulmonary fibrosis in scleroderma.

OBJECTIVE

This review focuses on the latest understanding of the molecular mechanisms underlying the complex interactions between intestine and liver bile acid signaling, gut microbiota, and their impact on whole-body lipid, glucose and energy metabolism.

RESULTS

Hepatic bile acid synthesis is tightly regulated by the bile acid negative feedback mechanisms. Modulating the enterohepatic bile acid signaling greatly impacts the whole-body metabolic homeostasis. Recently, a positive feedback mechanism through intestine farnesoid X receptor (FXR) antagonism has been proposed to link gut microbiota to the regulation of bile acid composition and pool size. Two studies identified intestine Diet1 and hepatic SHP-2 as novel regulators of CYP7A1 and bile acid synthesis through the gut-liver FXR-<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>/19-FGF receptor four signaling axis. New evidence suggests that enhancing bile acid signaling in the distal ileum and colon contributes to the metabolic benefits of bile acid sequestrants and bariatric surgery.

CONCLUSIONS

Small-molecule ligands that target TGR5 and FXR have shown promise in treating various metabolic and inflammation-related human diseases. New insights into the mechanisms underlying the bariatric surgery and bile acid sequestrant treatment suggest that targeting the enterohepatic circulation to modulate gut-liver bile acid signaling, incretin production and microbiota represents a new strategy to treat obesity and type 2 diabetes.

The concentration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic <em>factors</em>. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with <em>15</em>% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic <em>factors</em> compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas.

Mice deficient in the organic solute transporter (Ost)-alpha subunit of the heteromeric organic solute and steroid transporter, Ostalpha-Ostbeta, were generated and were found to be viable and fertile but exhibited small intestinal hypertrophy and <em>growth</em> retardation. Bile acid pool size and serum levels were decreased by more than 60% in Ostalpha-/- mice, whereas fecal bile acid excretion was unchanged, suggesting a defect in intestinal bile acid absorption. In support of this hypothesis, when [3H]taurocholic acid or [3H]estrone 3-sulfate were administered into the ileal lumen, absorption was lower in Ostalpha-/- mice. Interestingly, serum cholesterol and triglyceride levels were also approximately <em>15</em>% lower in Ostalpha-/- mice, an effect that may be related to the impaired intestinal bile acid absorption. After intraperitoneal administration of [3H]estrone 3-sulfate or [3H]dehydroepiandrosterone sulfate, Ostalpha-/- mice had higher levels of radioactivity in their liver and urinary bladder and less in the duodenum, indicating altered hepatic, renal, and intestinal disposition. Loss of Ostalpha was associated with compensatory changes in the expression of several genes involved in bile acid homeostasis, including an increase in the multidrug resistance-associated protein 3, (Mrp3)/Abcc3, an alternate basolateral bile acid export pump, and a decrease in cholesterol 7alpha-hydroxylase, Cyp7a1, the rate-limiting enzyme in bile acid synthesis. The latter finding may be explained by increased ileal expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>), a negative regulator of hepatic Cyp7a1 transcription. Overall, these findings provide direct support for the hypothesis that Ostalpha-Ostbeta is a major basolateral transporter of bile acids and conjugated steroids in the intestine, kidney, and liver.

Cytochrome P450 enzymes of the 4A family (CYP4A) convert arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) in blood vessels of several vascular beds. The present study examined the effects of inhibiting the formation of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016) on the mitogenic response of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in human umbilical vein endothelial cells (HUVECs) in vitro, and on <em>growth</em> <em>factor</em>-induced angiogenesis in the cornea of rats in vivo. HET0016 (10 micromol/L and 20 microg, respectively) abolished the mitogenic response to VEGF in HUVECs and the angiogenic response to VEGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> in vivo by 80 to 90% (P < 0.001). Dibromododecenyl methylsulfonimide (DDMS), a structurally and mechanistically different inhibitor of 20-HETE synthesis, also abolished angiogenic responses when tested with VEGF. Additionally, administration of the stable 20-HETE agonist, 20-hydroxyeicosa-6(Z) <em>15</em>(Z)-dienoic acid (WIT003) induced mitogenesis in HUVECs and angiogenesis in the rat cornea in vivo. We studied the ability of HET0016 to alter the angiogenic response in the rat cornea to human glioblastoma cancer cells (U251). When administered locally into the cornea, HET0016 (20 microg) reduced the angiogenic response to U251 cancer cells by 70%. These results suggest that a product of CYP4A product, possibly 20-HETE, plays a critical role in the regulation of angiogenesis and may provide a useful target for reduction of pathological angiogenesis.

We recently reported <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥<em>15</em> FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a co<em>factor</em> of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by <em>15</em>-20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-<em>15</em> and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH(2)-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal <em>growth</em> <em>factor</em>-stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance.

The expression of the gene encoding the facilitated glucose transporter (GT) protein was studied in <em>fibroblast</em> cell lines. Addition of <em>15</em>% calf serum to confluent BALB/c3T3, NIH3T3, or Rat-2 cells rapidly induced a 5-10-fold increase in GT mRNA, as determined by hybridization of size-fractionated total RNA to a rat brain GT cDNA. The rise in GT mRNA was maximal at 3-4 h after stimulation, and then returned to basal values by 16 h. The serum-stimulated increase in GT mRNA was not blocked by the protein synthesis inhibitors cycloheximide (10 micrograms/ml) or anisomycin (100 microM). In BALB/c3T3 cells, <em>fibroblast</em> <em>growth</em> <em>factor</em> (100 ng/ml), platelet-derived <em>growth</em> <em>factor</em> (5 units/ml), and epidermal <em>growth</em> <em>factor</em> (40 ng/ml) stimulated GT mRNA accumulation, although, when added individually, none of these <em>growth</em> <em>factors</em> increased DNA synthesis. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates the enzyme protein kinase C, also caused GT mRNA accumulation in BALB/c3T3 and NIH3T3 cells. Prolonged pretreatment of cells with TPA abolished the response to TPA but not <em>fibroblast</em> <em>growth</em> <em>factor</em>. The involvement of GT gene transcription was assessed by the nuclear run-on technique. Treatment of NIH3T3 cells with serum increased transcription at least 10-20-fold by 30 min and returned to near basal levels by 2 h. This rapid activation paralleled that of the c-fos gene, but preceded the increase in c-myc gene transcription. These data indicate the following: 1) serum <em>growth</em> <em>factors</em> increase glucose transporter mRNA levels by a process not requiring intermediary new protein synthesis and clearly dissociable from mitogenesis, 2) the changes in GT mRNA are preceded by a rapid and transient activation of GT gene transcription, and 3) there exist protein kinase C-dependent and independent pathways for regulation of GT gene expression.

To initiate a study of the role of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family in mammalian development, we have isolated cDNAs encoding four mouse FGF family members, aFGF, bFGF, kFGF, and FGF-5. This was achieved by a process that circumvents the use of cDNA libraries: for each family member, a cDNA fragment containing the conserved portion of the coding region was amplified from a pool of embryonic and teratocarcinoma cell cDNAs using the polymerase chain reaction (PCR) and cloned; the remaining coding sequences 5' and 3' to the conserved region were cloned using the RACE method. The cDNA clones obtained were used as probes to analyze the expression of these genes at the RNA level in teratocarcinoma cells and embryos at 10.5 to 17.5 days of gestation. Fgfk appears to be specific to undifferentiated teratocarcinoma stem cells. Fgf5 transcripts were detected at every stage and in every tissue tested, but showed a dramatic <em>15</em>-fold increase in abundance as teratocarcinoma stem cells differentiated to simple embryoid bodies. Fgfb expression showed the greatest tissue-specific variability in abundance, with the highest levels detected in the developing limbs and tail. Fgfa showed the least variable pattern of expression, with transcripts detected at roughly equivalent levels in almost all samples analyzed. On the basis of these data we speculate on some possible roles that the different FGF family members may play in the developing embryo.

OBJECTIVE

HIV causes inflammation that can be at least partially corrected by HAART. To determine the qualitative and quantitative nature of cytokine perturbation, we compared cytokine patterns in three HIV clinical groups, including HAART responders (HAART), untreated HIV noncontrollers, and HIV-uninfected (NEG).

METHODS

Multiplex assays were used to measure 32 cytokines in a cross-sectional study of participants in the Women's Interagency HIV Study. Participants from three groups were included: HAART (n = 17), noncontrollers (n = 14), and HIV NEG (n = 17).

RESULTS

Several cytokines and chemokines showed significant differences between noncontrollers and NEG participants, including elevated interferon gamma-induced 10 (IP-10) and tumor necrosis <em>factor</em>-α (TNF-α) and decreased interleukin-12(p40) [IL-12(p40)], IL-<em>15</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) in noncontroller participants. Biomarker levels among HAART women more closely resembled the NEG, with the exception of TNF-α and FGF-2. Secondary analyses of the combined HAART and noncontroller groups revealed that IP-10 showed a strong, positive correlation with viral load and negative correlation with CD4(+) T-cell counts. The <em>growth</em> <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, and FGF-2 all showed a positive correlation with increased CD4(+) T-cell counts.

CONCLUSIONS

Untreated, progressive HIV infection was associated with decreased serum levels of cytokines important in T-cell homeostasis (IL-<em>15</em>) and T-cell phenotype determination (IL-12), and increased levels of innate inflammatory mediators such as IP-10 and TNF-α. HAART was associated with cytokine profiles that more closely resembled those of HIV-uninfected women. The distinctive pattern of cytokine levels in the three study groups may provide insights into HIV pathogenesis, and responses to therapy.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription <em>factor</em> that plays an important role in the regulation of insulin sensitivity and lipid metabolism. Evidence shows that PPAR-gamma agonists also ameliorate renal fibrotic lesions in both diabetic nephropathy and nondiabetic chronic kidney disease. However, little is known about the mechanism underlying their antifibrotic action. This study demonstrated that PPAR-gamma agonists could exert their actions by inducing antifibrotic hepatocyte <em>growth</em> <em>factor</em> (HGF) expression. Incubation of mesangial cells with natural or synthetic PPAR-gamma agonists <em>15</em>-deoxy-Delta12,14-prostaglandin J2 (<em>15</em>d-PGJ2) or troglitazone and ciglitazone suppressed TGF-beta1-mediated alpha-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression. PPAR-gamma agonists also induced HGF mRNA expression and protein secretion. Transfection studies revealed that <em>15</em>d-PGJ2 stimulated HGF gene promoter activity, which was dependent on the presence of a novel peroxisome proliferator response element. Treatment of mesangial cells with <em>15</em>d-PGJ2 induced the binding of PPAR-gamma to the peroxisome proliferator response element in the HGF promoter region. PPAR-gamma agonists also activated c-met receptor tyrosine phosphorylation, induced Smad transcriptional co-repressor TG-interacting <em>factor</em> expression, and blocked TGF-beta/Smad-mediated gene transcription in mesangial cells. Furthermore, ablation of c-met receptor through the LoxP-Cre system in mesangial cells abolished the antifibrotic effect of <em>15</em>d-PGJ2. PPAR-gamma activation also induced HGF expression in renal interstitial <em>fibroblasts</em> and repressed TGF-beta1-mediated myofibroblast activation. Both HGF and <em>15</em>d-PGJ2 attenuated Smad nuclear translocation in response to TGF-beta1 stimulation in renal <em>fibroblasts</em>. Together, these findings suggest that HGF may act as a downstream effector that mediates the antifibrotic action of PPAR-gamma agonists.

Bile acids activate farnesoid X receptor (FXR) and G protein-coupled bile acid receptor-1 (aka Takeda G protein-coupled receptor-5 [TGR5]) to regulate bile acid metabolism and glucose and insulin sensitivity. FXR and TGR5 are coexpressed in the enteroendocrine L cells, but their roles in integrated regulation of metabolism are not completely understood. We reported recently that activation of FXR induces TGR5 to stimulate glucagon-like peptide-1 (GLP-1) secretion to improve insulin sensitivity and hepatic metabolism. In this study, we used the intestine-restricted FXR agonist fexaramine (FEX) to study the effect of activation of intestinal FXR on the gut microbiome, bile acid metabolism, and FXR and TGR5 signaling. The current study revealed that FEX markedly increased taurolithocholic acid, increased secretion of <em>fibroblast</em> <em>growth</em> <em>factors</em> <em>15</em> and 21 and GLP-1, improved insulin and glucose tolerance, and promoted white adipose tissue browning in mice. Analysis of 16S ribosomal RNA sequences of the gut microbiome identified the FEX-induced and lithocholic acid-producing bacteria Acetatifactor and Bacteroides. Antibiotic treatment completely reversed the FEX-induced metabolic phenotypes and inhibited taurolithocholic acid synthesis, adipose tissue browning, and liver bile acid synthesis gene expression but further increased intestinal FXR target gene expression. FEX treatment effectively improved lipid profiles, increased GLP-1 secretion, improved glucose and insulin tolerance, and promoted adipose tissue browning, while antibiotic treatment reversed the beneficial metabolic effects of FEX in obese and diabetic mice.

CONCLUSIONS

This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP-1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

OBJECTIVE

In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT.

METHODS

Endobronchial biopsies from current smokers (CS; n = 17) and ex-smokers with COPD (ES; n = 15), smokers with normal lung function (NS; n = 16) and never-smoking control subjects (NC; n = 15) were stained for the EMT markers, S100A4, vimentin, epidermal growth factor receptor and matrix metalloproteinase-9.

RESULTS

Compared with NC, there was significant Rbm fragmentation in the CS, ES and NS groups, which was positively associated with smoking history in subjects with COPD. Staining for basal epithelial S100A4, epithelial epidermal growth factor receptor and matrix metalloproteinase-9 in cells within Rbm clefts, and for S100A4 in Rbm cells, was increased in the CS, NS and ES groups compared with the NC group. There was also increased Rbm cell S100A4 staining in the CS group compared with the ES and NS groups. Basal epithelial cell staining for S100A4 was inversely correlated with airflow limitation. Double staining for both S100A4 and vimentin further strengthened the likelihood that these changes represented active EMT.

CONCLUSIONS

This is the first detailed description of fragmentation and cellularity of the Rbm in smokers, which were most marked in subjects with COPD. The data are consistent with active EMT in these subjects.

While it has long been recognized that bile acids are essential for solubilizing lipophilic nutrients in the small intestine, the discovery in 1999 that bile acids serve as ligands for the nuclear receptor farnesoid X receptor (FXR) opened the floodgates in terms of characterizing their actions as selective signaling molecules. Bile acids act on FXR in ileal enterocytes to induce the expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>15</em>/19, an atypical FGF that functions as a hormone. FGF<em>15</em>/19 subsequently acts on a cell surface receptor complex in hepatocytes to repress bile acid synthesis and gluconeogenesis, and to stimulate glycogen and protein synthesis. FGF<em>15</em>/19 also stimulates gallbladder filling. Thus, the bile acid-FXR-FGF<em>15</em>/19 signaling pathway regulates diverse aspects of the postprandial enterohepatic response. Pharmacologically, this endocrine pathway provides exciting new opportunities for treating metabolic disease and bile acid-related disorders such as primary biliary cirrhosis and bile acid diarrhea. Both FXR agonists and FGF19 analogs are currently in clinical trials.

Pbx1 is a homeodomain transcription <em>factor</em> that has the ability to form heterodimers with homeodomain proteins encoded by the homeotic selector (Hox) gene complexes and increase their DNA-binding affinity and specificity. A current hypothesis proposes that interactions with Pbx1 are necessary for Hox proteins to regulate downstream target genes that in turn control <em>growth</em>, differentiation and morphogenesis during development. In pre B cell leukemias containing the t(1;19) chromosome translocation, Pbx1 is converted into a strong transactivator by fusion to the activation domain of the bHLH transcription <em>factor</em> E2A. The E2A-Pbx1 fusion protein should therefore activate transcription of genes normally regulated by Pbx1. We have used the subtractive process of representational difference analysis to identify targets of E2A-Pbx1. We show that E2A-Pbx1 can directly activate transcription of a novel member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family of intercellular signalling molecules, FGF-<em>15</em>. The FGF-<em>15</em> gene is expressed in a regionally restricted pattern in the developing nervous system, suggesting that FGF-<em>15</em> may play an important role in regulating cell division and patterning within specific regions of the embryonic brain, spinal cord and sensory organs.

OBJECTIVE

The fibroblast growth factor receptor (FGFR)-3 fusion genes have been recently demonstrated in a subset of non-small cell lung cancer (NSCLC). To aid in identification and treatment of these patients, we examined the frequency, clinicopathologic characteristics, and treatment outcomes of patients who had NSCLC with or without FGFR fusions.

METHODS

Fourteen known FGFR fusion variants, including FGFR1, FGFR2, and FGFR3, were detected by RT-PCR and verified by direct sequencing in 1,328 patients with NSCLC. All patients were also analyzed for mutations in EGFR, KRAS, HER2, BRAF, ALK, RET, and ROS1. Clinical characteristics, including age, sex, smoking status, stage, subtypes of lung adenocarcinoma, relapse-free survival, and overall survival, were collected.

RESULTS

Of 1,328 tumors screened, two (0.2%) were BAG4-FGFR1 fusion and 15 (1.1%) were FGFR3-TACC3 fusion. Six of 1,016 patients with lung adenocarcinoma were FGFR3-TACC3 fusions and 11 of 312 lung squamous cell carcinoma harbored BAG4-FGFR1 or FGFR3-TACC3 fusions. Compared with the FGFR fusion-negative group, patients with FGFR fusions were more likely to be smokers (94.1%, 16 of 17 patients, P < 0.001), significantly associated with larger tumor (>3 cm; 88.2%, 15 of 17 patients, P < 0.001) and with a tendency to be more poorly differentiated (53.9%, nine of 17 patients, P = 0.095).

CONCLUSIONS

FGFR fusions define a molecular subset of NSCLC with distinct clinical characteristics. FGFR is a druggable target and patients with FGFR fusions may benefit from FGFR-targeted therapy, which needs further clinical investigation.

The gastrointestinal epithelium produces a wide variety of peptides which may contribute to protection from injury as well as repair after injury occurs. Restitution, the initial phase of mucosal repair, is accomplished by rapid migration of the epithelium to re-establish surface epithelial continuity. A wide variety of <em>growth</em> <em>factors</em> and cytokines, which are produced both by the epithelium itself and by lamina propria cell populations, promote restitution in models of epithelial injury. These include members of the epidermal <em>growth</em> <em>factor</em> (EGF)/transforming <em>growth</em> <em>factor</em> (TGF)alpha, and the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) families, as well as a variety of cytokines (interleukin [IL]-1, IL-2, IL-4, IL-<em>15</em>, and interferon gamma) which interact with their cognate receptors on the intestinal epithelial basolateral surface. These <em>growth</em> <em>factors</em> and cytokines appear to promote restitution through a TGF beta-dependent pathway and act to both enhance expression of TGF beta and to entrance its bioactivation. In contrast, trefoil peptides, members of a recently recognized family of small proteins produced by goblet cells, both protect the epithelium and promote restitution following secretion onto the apical surface through mechanisms distinct from those peptides acting through TGF beta. Thus, rapid repair after epithelial injury is achieved through complementary mechanisms acting at the basolateral and apical surfaces of the epithelium.

Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor gene, egl-<em>15</em>, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-<em>15</em> alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-<em>15</em> RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-<em>15</em>(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.

Increased expression of transforming <em>growth</em> <em>factor</em> (TGF)-beta(1) and tumor necrosis <em>factor</em> (TNF)-alpha are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-alpha upregulates TGF-beta(1) expression in primary mouse lung <em>fibroblasts</em> (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-alpha upregulation of TGF-beta(1) in both primary MLFs and the Swiss 3T3 <em>fibroblast</em> cell line. Treatment of <em>fibroblasts</em> with TNF-alpha resulted in a significant increase in TGF-beta(1) protein as measured by ELISA. The increase in protein was preceded by a 200-400% increase in TGF-beta(1) mRNA detected by quantitative, real-time, reverse transcriptase-polymerase chain reaction. Western blot analysis showed that TNF-alpha activated the extracellular signal-regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-alpha induction of TGF-beta(1) mRNA and protein. mRNA stability experiments showed that TNF-alpha increased the half-life of TGF-beta(1) mRNA to more than 24 h compared with approximately <em>15</em> h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-beta(1) mRNA stabilization in Swiss 3T3 <em>fibroblasts</em>. These results indicate that TNF-alpha activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-beta(1) production in <em>fibroblasts</em>, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-beta(1) transcript.

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a <em>15</em>- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two <em>growth</em> <em>factors</em> of the FGF family.

Graves' disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like <em>growth</em> <em>factor</em> receptor-1 (IGF-1R)-bearing <em>fibroblasts</em> overpopulate connective tissues in GD. IGF-1R on <em>fibroblasts</em>, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 +/- 4% (mean +/- SE; n = 33) in patients with GD compared with <em>15</em> +/- 3% (n = 21; p < 10(-8)) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.

BACKGROUND

X-Linked hypophosphatemia (XLH) is characterized by renal phosphate wasting, with inappropriately low or normal serum 1,25-dihydroxyvitamin D concentrations causing rickets and osteomalacia. Mutations in PHEX result in increased fibroblast growth factor 23 (FGF23) expression, elevating circulating FGF23 concentrations. Treating XLH with phosphate and calcitriol may further increase FGF23 concentrations, based on in vitro and in vivo models.

OBJECTIVE

The aim of the study was to investigate whether current standard XLH therapies increase circulating FGF23 concentrations.

METHODS

We conducted a prospective observational study of XLH subjects during routine clinical management at two tertiary referral centers.

METHODS

The study included 10 XLH patients (seven children, three adults; age, 2-30 yr) initiating therapy and five XLH patients (age, 18-41 yr) electing not to undergo therapy.

METHODS

Oral calcitriol and phosphate were administered.

METHODS

We measured circulating intact FGF23 concentrations.

RESULTS

Baseline circulating FGF23 concentrations were elevated in 14 of 15 subjects, increasing after treatment in most subjects. Follow-up was 14.4 +/- 11.7 months (treatment cohort) and 25 +/- 32 months (nontreatment cohort). FGF23 concentrations increased 132.7 +/- 202.4% from pretreatment to peak during therapy but did not change significantly over time in the nontreatment cohort. FGF23 concentrations were related to phosphate doses (P = 0.04) and nonsignificantly to calcitriol doses (P = 0.06).

CONCLUSIONS

Treating XLH with phosphate and calcitriol was associated with concurrent increases in circulating FGF23 concentrations, which may diminish therapeutic effect or contribute to complications of therapy. Because it is unknown whether the degree of FGF23 elevation correlates with disease severity in XLH, further study is needed to determine whether adjusting therapy to minimize effects on FGF23 concentration is warranted.

Basic and clinical research have shown the efficacy of various cellular mediators (bone morphogenetic proteins, interleukins, angiogenic <em>growth</em> <em>factors</em>) in healing bone defects. The potential application of these <em>growth</em> and differentiation <em>factors</em> to other musculoskeletal conditions, including osteonecrosis of the femoral head, only recently has been explored. Osteonecrosis is a disease of unknown pathogenesis that usually progresses to hip joint destruction necessitating total hip arthroplasty. The pathology involves ischemic events followed by death of bone and marrow elements. A process of repair then is initiated, but unless the lesion is small (less than <em>15</em>% of the femoral head involved), this repair process is usually ineffective. The net result is weakening of subchondral bone with subsequent collapse of the articular surface. Because the results of hip arthroplasty in patients with osteonecrosis are relatively poor, much focus has been on modalities aimed at femoral head preservation. The surgical alternatives may include core decompression, osteotomy, nonvascularized, and vascularized bone grafting, which might be enhanced with the use of <em>growth</em> and differentiation <em>factors</em>. At least three of these <em>factors</em> are potential candidates as therapeutic modalities: cytokines (such as interleukins, tumor necrosis <em>factors</em>, and signaling molecules such as <em>fibroblast</em> <em>growth</em> <em>factors</em>, platelet derived <em>growth</em> <em>factors</em>, insulinlike <em>growth</em> <em>factors</em>, and transforming <em>growth</em> <em>factor</em> betas), bone morphogenetic proteins, and angiogenic <em>factors</em>. Despite considerable effort, evaluation of these <em>growth</em> and differentiation <em>factors</em> has been hampered by the lack of an animal model that adequately simulates the pathology of osteonecrosis in humans. Therefore, investigators have attempted to model certain aspects of the disease process. Recently, several investigators have attempted to mimic osteonecrosis in the femoral head of large mammals by combinations of devascularization, freezing, osteotomy of the femoral neck, or creation of a head defect. Results from some of these studies have confirmed the potential for <em>growth</em> and differentiation <em>factors</em> to effect more rapid healing and filling of defects with biomechanically competent and viable bone. The application of this therapy shows promise, and clinical studies on efficacy and safety are ongoing.