The <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> gene FGF<em>19</em> has previously been reported to be amplified in several cancer types and encodes for a key autocrine signaler known to promote tumorigenic <em>growth</em>. Thus, it is imperative to understand which cancers are oncogenically addicted to FGF<em>19</em> amplification as well as the role it serves in these cancer types. We report for the first time high FGF<em>19</em> amplification in head and neck squamous cell carcinomas (HNSCC), which is associated with increased autocrine secretion of FGF<em>19</em> and poor patient outcome in HNSCC. FGF<em>19</em> amplification corresponded with constitutive activation of FGF receptor 4 (FGFR4)-dependent ERK/AKT-p70S6K-S6 signaling activation in HNSCC cells, and addition of human recombinant FGF<em>19</em> could promote cell proliferation and soft agar colony formation in HNSCC cells with low FGF<em>19</em> expression through activation of FGFR4 and downstream signaling cascades. In contrast, FGF<em>19</em> knockout counteracts the observed effects in HNSCC cells carrying high endogenous FGF<em>19</em>, with knockout of FGF<em>19</em> significantly suppressing tumor <em>growth</em> in an orthotopic mouse model of HNSCC. Collectively, this study demonstrates that FGF<em>19</em> gene amplification corresponds with an increased dependency upon FGF<em>19</em>/FGFR4 autocrine signaling in HNSCC, revealing a therapeutic target for this cancer type.

BACKGROUND

We analyzed a large set of EGFR-mutated (EGFR+) NSCLC to identify and characterize cases with co-occurring kinase fusions as potential resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs).

METHODS

EGFR+ (del <em>19</em>, L858R, G7<em>19</em>X, S768I, L851Q) NSCLC clinical samples (formalin-fixed paraffin-embedded tumor and blood) were analyzed for the presence of receptor tyrosine kinase (RTK) and BRAF fusions. Treatment history and response were obtained from provided pathology reports and treating clinicians.

RESULTS

Clinical samples from 3505 unique EGFR+ NSCLCs were identified from June 2012 to October 2017. A total of 31 EGFR+ cases had concurrent kinase fusions detected: 10 (32%) BRAF, 7 (23%) ALK receptor tyrosine kinase (ALK), 6 (<em>19</em>%) ret proto-oncogene (RET), 6 (<em>19</em>%) fibroblast growth factor receptor 3 (FGFR3), 1 (3.2%) EGFR, and 1 (3.2%) neurotrophic receptor tyrosine kinase 1 (NTRK1), including two novel fusions (SALL2-BRAF and PLEKHA7-ALK). Twenty-seven of 31 patients had either a known history of EGFR+ NSCLC diagnosis or prior treatment with an EGFR TKI before the fusion+ sample was collected. Twelve of the 27 patients had paired pre-treatment samples where the fusion was not present before treatment with an EGFR TKI. Multiple patients treated with combination therapy targeting EGFR and the acquired fusion had clinical benefit, including one patient with osimertinib resistance due to an acquired PLEKHA7-ALK fusion achieving a durable partial response with combination of full-dose osimertinib and alectinib.

CONCLUSIONS

RTK and BRAF fusions are rare but potentially druggable resistance mechanisms to EGFR TKIs. Detection of RTK and BRAF fusions should be part of comprehensive profiling panels to determine resistance to EGFR TKIs and direct appropriate combination therapeutic strategies.

We first studied expression of neurofibromin by immunohistochemistry in scars obtained from operations involving areas of healing wounds. The results demonstrated increased immunoreactivity for neurofibromin in the fibroblastic cell population of the lesions when compared with <em>fibroblasts</em> of apparently healthy perilesional skin, or those of intact control skin. Furthermore, dermal <em>fibroblasts</em> of <em>19</em> and 34 wk-old fetuses displayed a clearly detectable immunosignal for neurofibromin. In vitro studies were designed to investigate the potential effects of selected <em>growth</em> <em>factors</em>--known to be operative in wound healing--on neurofibromin mRNA steady-state levels in cultured <em>fibroblasts</em>. Northern transfer analyses revealed that different isoforms of platelet derived <em>growth</em> <em>factor</em> (PDGF) exerted selective effects on the neurofibromin mRNA levels: PDGF isoform AB elevated neurofibromin mRNA levels in a concentration-dependent manner when concentrations of 0.1, 1, 10, and 30 ng per ml were used. The maximal upregulatory effect of PDGF BB was reached at a concentration of 1 ng per ml. In contrast, PDGF AA did not alter the steady-state levels of neurofibromin mRNA. As estimated by RNase protection assay, transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) upregulated neurofibromin gene expression when concentrations of 0.5 and 5 ng per ml were used. Reverse transcription followed by polymerase chain reaction did not detect apparent alterations in the ratio of type I/type II neurofibromin isoforms in PDGF- or TGF-beta1-treated cultures. Taken together, our results suggest that expression of tumor suppressor protein neurofibromin is upregulated in response to skin injury, and that this upregulation can be mediated through PDGF and TGF-beta.

OBJECTIVE

Formerly obese patients having undergone Roux-en-Y gastric bypass (RYGB) display both an accelerated digestion and absorption of carbohydrate and an increased plasma glucose clearance rate after meal ingestion. How RYGB effects postprandial kinetics of dietary lipids has yet not been investigated.

METHODS

Plasma triglyceride (TG), apoB48, total apoB, bile acids (BA), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), and cholecystokinin (CCK) were measured in post-absorptive conditions and over 4-h following the ingestion of a mixed test meal in a cross-sectional, pilot study involving 11 formerly obese female patients 33.8 ± 16.4 months after RYGB surgery and in 11 weight- and age-matched female control participants.

RESULTS

Compared to controls, RYGB patients had faster (254 ± 14 vs. 327 ± 7 min, p < 0.05) and lower (0.14 ± 0.04 vs. 0.35 ± 0.07 mM, p < 0.05) peak TG responses, but their peak apoB48 responses tended to be higher (2692 ± 336 vs. 1841 ± 228 ng/ml, p = 0.09). Their postprandial total BA concentrations were significantly increased and peaked earlier after meal ingestion than in controls. Their FGF<em>19</em> and CCK concentrations also peaked earlier and to a higher value.

CONCLUSIONS

The early postprandial apoB48 and BA responses indicate that RYGB accelerated the rate of dietary lipid absorption. The lower postprandial peak TG strongly suggests that the RYGB simultaneously increased the clearance of TG-rich lipoproteins.

BACKGROUND

NCT01891591.

Somatic activating <em>fibroblast</em> <em>growth</em> <em>factor</em> 3 (FGFR3) mutations in human skin can cause seborrheic keratoses, one of the most frequent skin tumors in man. However, details of the involved mechanisms remain elusive. We analyzed 65 acanthotic seborrheic keratoses with varying vertical diameters for FGFR3 mutations using a SNaPshot multiplex assay. Immunohistochemistry was performed for Ki-67, bcl-2 and FGFR3 protein in all seborrheic keratoses and <em>19</em> normal skin samples. FGFR3 mutations were detected in 37 of 65 seborrheic keratoses (57%). These mutations were found both in flat (initial) and thick seborrheic keratoses. FGFR3 mutations were significantly associated with increased age and localization on the head and neck (P<0.01). Ki-67 expression was significantly higher in seborrheic keratoses than in normal epidermis independent of the FGFR3 status (P<0.001). Furthermore, FGFR3 mutations were associated with an increased expression of bcl-2 and FGFR3 protein (P<0.05). Our results indicate that FGFR3 mutations can occur early in the pathogenesis of at least a subset of seborrheic keratoses. Increased age appears to be a risk <em>factor</em> for these mutations. The preferential occurrence of FGFR3 mutations in seborrheic keratoses of the head and neck suggests a causative role for cumulative lifetime ultraviolet light exposure.

Although insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Serum of acutely fasted rats contained reduced amount of IGF-I (72+/-16 ng/ml) and showed about 75% reduced ability to stimulate collagen and DNA synthesis in confluent human skin <em>fibroblasts</em> in comparison to the effect of control rat serum (IGF-I, 168+/-<em>19</em> ng/ml). An addition of IGF-I (at least 40 ng/ml) to fasted rat serum restored its mitogenic activity but could not restore its ability to stimulate collagen biosynthesis to control values during 24 h of incubation. However, when the cells were incubated in fasted rat serum supplemented with 40 ng/ml of IGF-I for 48 h, collagen biosynthesis was restored to control values. It suggests that the stimulatory role of IGF-I in collagen biosynthesis undergo indirectly. We considered pyrroline-5-carboxylate (P5C) as a candidate to play a direct role in this process. Since IGF-I and P5C are known to be decreased in serum of fasted rats it seems that the action of IGF-I on collagen biosynthesis may involve participation of P5C. We have found that serum of fasted rats (showing low level of P5C) supplemented with 1 mmol/l P5C induced collagen biosynthesis in confluent human skin <em>fibroblasts</em> during 24 h to control values. Supporting evidence comes from the experiment showing stimulatory action of P5C on collagen biosynthesis in <em>fibroblasts</em> cultured in serum-free medium. Our results postulate potential role of P5C in regulation of collagen biosynthesis and indicate participation of this molecule in the pathway of IGF-I action in this process.

De novo bile acid synthesis in the liver needs to be tightly regulated in order to maintain optimal bile flow and prevent cholestasis. In the liver, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) regulates bile acid synthesis by down-regulating messenger RNA levels of cytochrome P450 7A1 (CYP7A1). FGF<em>19</em> acts through <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4), and beta-Klotho has recently been recognized as a modulator of FGFR4 activity. However, its precise mechanism of action has not been thoroughly described. We show here that beta-Klotho is an endoplasmic reticulum-resident protein that affects the cellular abundance of different FGFR4 glycoforms. beta-Klotho binds and directs the core glycoform of FGFR4 to the proteasome, and it allows only a terminal glycoform to reach the plasma membrane. Only the terminal FGFR4 glycoform is phosphorylated upon FGF<em>19</em> treatment of HepG2 cells, and this shows that only fully glycosylated FGFR4 is active in CYP7A1 down-regulation.

CONCLUSIONS

beta-Klotho enhances FGF<em>19</em> signaling by binding the inactive, core-glycosylated FGFR4 and preventing it from reaching the surface. These results indicate that beta-Klotho is an indirect regulator of FGFR4, whereas glycosylation is the master switch for FGF<em>19</em> activity and regulation of bile acid synthesis.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a recently described heparin-binding mitogen for <em>fibroblasts</em> and smooth muscle cells. The aim of this study was to investigate the production of CTGF by human uterine tissues using immunohistochemical and Northern blotting analyses. For immunohistochemistry, formalin-fixed human proliferative (n = 5), early secretory (n = 5; days 15-<em>19</em>), mid-secretory (n = 5; days 20-23), late secretory (n = 5; days 24-28) endometrial, and decidual (n = 5) tissues were stained using a highly specific affinity-purified polyclonal antibody raised against residues 81-94 of human CTGF. Myometrial (n = 5) and leiomyoma (n = 5) tissues were also used for CTGF immunochemistry. In proliferative endometrium, epithelial and vascular endothelial cells showed strong CTGF immunoreactivity, whereas stromal cells were negative or only weakly positive for the CTGF protein. Throughout the entire secretory stage, CTGF was detected in epithelial and endothelial cells of endometrium. Stromal cells showed strong immunoreactivity to CTGF only in oedematous areas for early and mid-secretory endometrium, and in decidualized regions of late secretory endometrium. During pregnancy, the decidual, epithelial and endothelial cells of the endometrium were all immunoreactive to CTGF. In myometrial and leiomyoma samples, CTGF immunoreactivity was found only in the endothelial cells. Northern blotting of mRNA from normal uterus (n = 2) or leiomyoma (n = 6) using a 320 bp human CTGF cDNA probe revealed a single 2.4 kb transcript. This study is the first to demonstrate CTGF gene expression and localization of its encoded protein in human uterine tissues. The cell- and cycle-specific localization of CTGF support a role for this molecule in regulating aspects of uterine cell <em>growth</em>, migration, and/or matrix production during the menstrual cycle and pregnancy.

Angiogenesis is essential in tissue <em>growth</em> and regeneration. There are several <em>factors</em> that are able to stimulate vascular endothelial cell <em>growth</em>, including platelet-derived <em>growth</em> <em>factor</em> (PDGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Disc herniation tissue (DHT) contains vascular in<em>growth</em>, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in <em>19</em> samples (38%) and PDGF immunopositive <em>fibroblasts</em> in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither <em>growth</em> <em>factor</em> was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in <em>fibroblasts</em> and in disc cells suggests that this <em>growth</em> <em>factor</em> regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.

OBJECTIVE

Increased colonic bile acids can cause chronic diarrhea. Bile acid diarrhea (BAD) is treatable by sequestrants, and may be secondary to ileal disease or primary BAD. It is underdiagnosed, partly because the selenium-75-homocholic acid taurine (SeHCAT) retention test is not available in many countries, and is underutilized in others. Serum 7α-hydroxy-4-cholesten-3-one (C4), a measure of bile acid synthesis, is available for diagnosis in specialist centers. Recently, deficiency of the ileal hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) has been shown in BAD. Our aim is to evaluate the diagnostic value of FGF<em>19</em> in a large and prospective group of patients with chronic diarrhea, previously investigated with C4.

METHODS

Patients undergoing routine investigation provided fasting blood samples. C4 was determined by high-performance liquid chromatography, and used to stratify two groups: group 1 (n=1<em>19</em>), consisting of patients with normal C4 (≤ 28 ng/ml), and group 2 (n=139), consisting of patients with high C4 (>28 ng/ml), including any of the possible causes of BAD. Serum FGF<em>19</em> was measured in stored samples by enzyme-linked immunosorbent assay.

RESULTS

FGF<em>19</em> and C4 were significantly inversely related (r(s)=-0.64, P<0.001). Patients with raised C4 had significantly lower median FGF<em>19</em> values. Both of these were more marked when secondary to ileal disease, in particular ileal resection, than in primary BAD. The sensitivity and specificity of FGF<em>19</em> at 145 pg/ml for detecting a C4 level >28 ng/ml were 58% and 79%, respectively. For C4 >60 ng/ml, these were 74% and 72%; on receiver-operating characteristic analysis, the area under the curve was 0.80 (95% confidence interval 0.74-0.87).

CONCLUSIONS

Serum FGF<em>19</em> could be developed as a simple blood test to increase the diagnostic rates of BAD.

MMP-<em>19</em> (also designated RASI) is a recently discovered member of a large family of zinc-dependent proteolytic enzymes, most of which have been implicated in cancer <em>growth</em> and metastasis. It differs from the others by its chromosomal location and structure and is expressed by endothelial and vascular smooth muscle cells in vivo. Our aim was to study the putative role of MMP-<em>19</em> in skin cancer. We also examined its regulation in keratinocyte cultures using quantitative TaqMan RT-PCR. Our results show that MMP-<em>19</em> can also be detected in stimulated keratinocytes by Northern and Western analyses. In wounds, it was found in keratinocytes outside the migrating area, while in BCC and SCC, it was present in the hyperproliferative (p63-positive), E-cadherin-negative epidermis at the tumor surface but downregulated in invasive cancer islands. Expression was also evident in endothelial cells of neoangiogenic regions and in occasional stromal <em>fibroblasts</em>. Of the 12 tested cytokines/<em>growth</em> <em>factors</em>, only TNF-alpha and PMA were able to stimulate the expression of MMP-<em>19</em> mRNA in primary keratinocytes. No MMP-<em>19</em> mRNA was detected by Northern analysis in cultured HaCaT or A5 cells or in an SCC cell line established from head-and-neck cancer. Our data suggest that, unlike most MMPs, MMP-<em>19</em> expression in the epidermis is downregulated during transformation and histologic dedifferentiation.

BACKGROUND

Juvenile nasopharyngeal angiofibroma (JNA) is a histologically benign, locally aggressive neoplasm of the nasopharynx that exclusively affects male adolescents. It is known to be sensitive to androgens, but there are likely intermediary cytokines and/or growth factors that mediate aggressive stromal cell proliferation and angiogenesis. Transforming growth factor beta1 (TGF-beta1) is a polypeptide that is secreted in an inactive form, cleaved to produce an active form, and then deactivated in the tissues. It activates fibroblast proliferation and is known to induce angiogenesis.

OBJECTIVE

To evaluate the presence of activated TGF-beta1 within the stroma of JNA specimens and to quantify the percentage of JNA specimens expressing the active growth factor.

METHODS

Immunohistochemical analysis was performed on 19 specimens of JNA using a unique antibody that identifies only the activated form of TGF-beta1. The percentage of cells staining positively for activated TGF-beta1 was determined semiquantitatively by visual methods.

RESULTS

Of 19 cases stained, all 19 (100%) showed strong positive staining (2 cases with 33%-66% of cells staining and 17 with 66%-100% of cells staining). Activated TGF-beta1 was identified in stromal cell nuclei and cytoplasm and in the endothelium of the capillaries within all specimens of JNA.

CONCLUSIONS

The localization of activated TGF-beta1 to the fibroblasts and endothelial cells within JNA tumors suggests that TGF-beta1 may play a role in the stromal cell proliferation and angiogenesis associated with JNA. Additional receptor studies and more quantitative methods of analysis are needed to further define the role of TGF-beta1 in the pathogenesis of JNA.

Early diagnosis of breast cancer is the key to extending survival of breast-cancer patients. We found that the concentrations of nipple fluid bFGF (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) was significantly increased in breast-cancer patients compared with concentrations in controls (1717 ng/L [SD 706] vs <em>19</em> ng/L [<em>19</em>]; Student's t test p=0.027). Measurement of bFGF in nipple fluid could be a useful diagnostic tool for breast cancer, and deserves further study.

Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon out<em>growth</em> and pathfinding. On the basis of their localized expression patterns, we hypothesized that bone morphogenetic proteins (BMPs), <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), and sonic hedgehog (Shh) may function as guidance cues for <em>growing</em> axons and/or may function as trophic <em>factors</em> once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow Embryonic Day 4 (E4) chick SAG explants for 24 h in the presence of purified proteins or beads soaked in proteins. The density of neurite out<em>growth</em> was quantified to determine effects on neurite out<em>growth</em>. Explants displayed enhanced neurite out<em>growth</em> when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF10, or FGF<em>19</em>. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with terminal dUTP nick-end labeling and immunostained with anti-phosphohistone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite out<em>growth</em> also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -10, and -<em>19</em> promoted asymmetric out<em>growth</em> from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon out<em>growth</em> of otic neurons.

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mouse <em>fibroblasts</em>. The transmembrane (TM) domain of E5 is not only critical for biological activity, it also regulates interactions with cellular targets including the platelet derived <em>growth</em> <em>factor</em> receptor (PDGF-R) and the 16-kDa subunit of the vacuolar proton ATPase (V-ATPase). In order to define the specific TM amino acids essential for E5 biological and biochemical activity, we performed scanning alanine mutagenesis on 25 of the 30 potential TM residues and genetically mapped discrete alpha-helical domains which separately regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to form oligomers. Alanine substitutions at positions 17, 21, and 24 (which lie on the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R activation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, 16, <em>19</em>, 22, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant proteins with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, 21, 24, and 25) did not, indicating that the latter helical face was critical for mediating receptor transphosphorylation. Interestingly, these "activation-defective" mutants segregated into two classes: 1) those that were unable to form dimers but that could still form higher order oligomers and transform cells, and 2) those that were defective for PDGF-R binding and were transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation. However, since several transformation-competent E5 mutants were defective for binding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (homodimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.

HePTP is a tyrosine specific protein phosphatase that is strongly expressed in activated T-cells. It was recently demonstrated that in transfected T-cells HePTP impairs TCR-mediated activation of the MAP-kinase family members ERK2 and p38 and it was suggested that both ERK and p38 MAP-kinases are substrates of HePTP. The HePTP gene has been mapped to human chromosome 1q32.1. Abnormalities in this region are frequently found in various hematopoietic malignancies. HePTP is highly expressed in acute myeloid leukemia and its expression in <em>fibroblasts</em> resulted in transformation. To address a possible involvement of HePTP in hematopoietic malignancies we sought to identify HePTP substrate(s) in leukemic cells. Using substrate trapping mutants we have identified the MAP-kinase ERK2 as a specific target of HePTP in the myelogenous leukemia cell line K562. Tyrosine phosphorylated ERK2, but not ERK1, p38, or JNK1, efficiently bound to catalytically inactive HePTP mutants in which the active site cysteine (HePTP-C/S) or the conserved aspartic acid residue (HePTP-D/A) had been exchanged for serine and alanine, respectively. Moreover, the interaction of ERK2 with HePTP trapping mutants was dependent on ERK2 tyrosine phosphorylation, indicating that HePTP is specifically targeted to activated ERK2. Using a deletion mutant of HePTP (HePTP-dLD), in which 14 amino acid residues within the N-terminus are missing, we show that regions outside the catalytic domain are also required for the interaction. Furthermore, overexpression of HePTP in K562 cells and <em>fibroblasts</em> interfered with PMA or <em>growth</em> <em>factor</em> induced MAP-kinase activation and HePTP efficiently dephosphorylated active ERK2 on the tyrosine residue in the activation loop in vitro. Together, these data identify ERK2 as a specific and direct target of HePTP and are consistent with a model in which HePTP negatively regulates ERK2 activity as part of a feedback mechanism. Oncogene (2000) <em>19</em>, 858 - 869.

The precursor for transforming <em>growth</em> <em>factor</em>-alpha, proTGF-alpha, is synthesized as an integral membrane glycoprotein with the mature TGF-alpha sequence located in the extracellular domain. Retrovirally transformed rat embryo <em>fibroblasts</em> (FeSV-Fre cells) expressing the endogenous proTGF-alpha gene release and accumulate in the medium mature TGF-alpha as well as a heterogeneous (17-<em>19</em> kDa) group of soluble, bioactive TGF-alpha precursor forms. These precursors correspond to the heterogeneously glycosylated extracellular domain of proTGF-alpha which is released from the membrane by proteolytic cleavage. They are designated mesoTGF-alpha to denote their intermediate position in the proTGF-alpha processing pathway. The nature of the carbohydrate linked to mesoTGF-alpha has been examined by treatment with glycosidases and the use of metabolic inhibitors of glycosylation. The results indicate that the TGF-alpha precursors from FeSV-Fre cells contain O-linked carbohydrate as well as sialylated N-linked carbohydrate. Heterogeneous N-linked glycosylation of an 11-kDa core polypeptide accounts for the heterogeneous nature of mesoTGF-alpha. MesoTGF-alpha released by cells treated with inhibitors of N-linked carbohydrate processing appears as a 17-kDa species. Treatment with these inhibitors does not alter significantly the production of mesoTGF-alpha or mature TGF-alpha by the cells. However, treatment of cells with an inhibitor of co-translational N-linked glycosylation, tunicamycin, reduces the accumulation of mesoTGF-alpha in the medium and blocks the production of mature TGF-alpha under conditions in which overall protein synthesis is only minimally affected. These findings suggest that the proTGF-alpha processing activity is limiting in FeSV-Fre cells and other transformed cells that accumulate mesoTGF-alpha in the medium and that proTGF-alpha processing depends on a component whose function may require N-linked glycosylation.

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like <em>growth</em> <em>factor</em> I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal <em>fibroblasts</em> and myoblasts. Myoblasts were derived from skeletal muscle and <em>fibroblasts</em> from skin explants removed from human fetuses delivered between 12 and <em>19</em> weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both <em>fibroblasts</em> and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For <em>fibroblast</em> cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal <em>fibroblasts</em>. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the <em>growth</em> stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.

Background and rationale for the study. FGF19/15 is a gut-derived hormone presumably governing bile acid (BA) synthesis and gallbladder (GB) refilling. FGF19 mRNA is present in human GB cholangiocytes (hGBECs); however, the physiological significance of GB-derived FGF19 remains unknown. We investigated whether hGBECs secrete FGF19 and the effects of cholecystectomy on serum FGF19 ([FGF19]s) and BA synthesis.

METHODS

FGF19 expression was assessed by qRT-PCRs and immunostaining in hGBECs and terminal ileum, and quantified in bile and serum by ELISA. Basal and BA (chenodexycholic acid, CDCA) induced FGF19 expression and secretion was analyzed in primary cultured hGBECs and GB-d1 cell line. Pre and postprandial serum changes in [FGF19]s, 7α-hydroxy-4-cholestene-3-one (C4, a marker of BA synthesis) and BA were evaluated in plasma of gallstone disease patients at baseline and after cholecystectomy.

RESULTS

FGF19 mRNA levels were ~250-fold higher in hGBECs compared to distal ileum. GB bile contained ~23-fold higher FGF19 levels compared to serum (p < 0.0001). CDCA induced dose-dependent expression and secretion of FGF19 in hGBECs and GB-d1 cells. Cholecystectomy increased plasma BA synthesis ≥ 2-fold (p < 0.0001), and altered the diurnal rhythm and significantly reduced [FGF19]s noon peak. BA serum levels, serum cholesterol and triglyceride content remained unchanged.

CONCLUSIONS

In conclusion human GB cholangiocytes constitutively express and secrete high levels of FGF19 in a process regulated by BA. Resection of this organ doubles BA synthesis concomitantly with changes in [FGF19]s. These findings suggest a potential connection between GB cholangiocytes-derived FGF19 and BA metabolism that could lead to metabolic dysregulation following cholecystectomy.

The present study aimed to investigate the actions of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF21 (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF21 caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF21-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by <em>19</em>% in FGF21 treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF21 maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF21 in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF21 with respect to promoting a lean phenotype. The observed differences in FGF21 sensitivity may relate to day length-induced changes in adipose tissue mass.

BACKGROUND

The highly vascular nature of renal cell carcinoma (RCC) suggests that angiogenesis inhibition may be therapeutic for patients with this disease. Thalidomide inhibits basic fibroblast growth factor and vascular endothelial growth factor (VEGF)-induced angiogenesis.

METHODS

In a pilot study, we evaluated the safety and efficacy of escalating doses of thalidomide in patients with progressive metastatic RCC (mRCC), measurable disease, and good organ function. Patients received oral thalidomide starting at 200 mg per day and increasing by 100-200 mg per day weekly until a target dose of 1200 mg per day was reached. Study endpoints were objective tumor response and toxicity.

RESULTS

Of the 20 patients enrolled, 19 were evaluable for response. Eighteen achieved the target dose. The most common, but reversible, toxicities were constipation, somnolence, and fatigue. Peripheral neuropathy was seen after prolonged therapy, necessitating dose reduction. Two patients achieved a partial response and nine had stable disease for a median of 14 months (range, 3-17 months). Median time to progression was 4.7 months (range, 0.7-31.3 months). Fifteen patients died (median survival, 18.1 months; 95% lower confidence bound 10.7). Survival was significantly longer in patients with higher hemoglobin level and longer time from first metastasis to start of thalidomide, but significantly shorter in patients with multiple organ involvement and previous treatments.

CONCLUSIONS

Thalidomide at this dose is associated with manageable acute toxicities but long-term dose-limiting neuropathy. Objective responses are rare in patients with mRCC and are characterized by delay in achieving maximum tumor reduction. Prolonged stable disease is seen in some patients, but the benefit of thalidomide, as well as other angiogenesis inhibitors, in that setting needs to be studied in controlled, randomized trials.

Diarrhea-associated hemolytic uremic syndrome (D+HUS) is characterized by endothelial injury and activation of inflammatory cytokines. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is an angiogenic peptide released in response to vascular damage. The plasma concentrations and urinary excretion of bFGF during the course of D+HUS were determined, in comparison with the levels of various inflammatory cytokines, and changes were correlated with clinical and laboratory features of the disease. Serial plasma and urine samples were collected from 31 children with D+HUS, during the acute (days 1 to 7 of hospitalization) and recovery (through day 60 after discharge from the hospital) phases of the disease. The patients were enrolled in the multicenter trial of SYNSORB Pk (SYNSORB Biotech, Calgary, Alberta, Canada) treatment for D+HUS. bFGF, interleukin-1alpha (IL-1alpha), IL-8, and tumor necrosis <em>factor</em>-alpha levels were determined with enzyme-linked immunosorbent assays. bFGF was detected in urine and plasma samples more frequently than were IL-1alpha, IL-8, and tumor necrosis <em>factor</em>-alpha. There was an acute increase in urinary bFGF excretion, which returned to normal during convalescence. Urinary excretion of bFGF during the acute phase was higher among patients who required dialysis, compared with those who did not (48.9 +/- 15.0 and 28.9 +/- 9.0 pg/ml, respectively; P < 0.05). Plasma bFGF concentrations were persistently elevated throughout the period of hospitalization and the follow-up period among patients with D+HUS. Urinary excretion and plasma levels of bFGF were comparable for the SYNSORB Pk-treated (n = <em>19</em>) and placebo-treated (n = 12) groups. Measurements of urinary and plasma concentrations of bFGF among patients with D+HUS may be useful indices for assessment of the severity of acute renal disease and the timing and adequacy of the systemic angiogenic process during early convalescence.

High level contamination by natural and industrial sources of the alkali earth metal, barium (Ba) has been identified in the ecosystems/workplaces that are associated with high incidence clustering of multiple sclerosis (MS) and other neurodegenerative diseases such as the transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis (ALS). Analyses of ecosystems supporting the most renowned MS clusters in Saskatchewan, Sardinia, Massachusetts, Colorado, Guam, NE Scotland demonstrated consistently elevated levels of Ba in soils (mean: 1428 ppm) and vegetation (mean: 74 ppm) in relation to mean levels of 345 and <em>19</em> ppm recorded in MS-free regions adjoining. The high levels of Ba stemmed from local quarrying for Ba ores and/or use of Ba in paper/foundry/welding/textile/oil and gas well related industries, as well as from the use of Ba as an atmospheric aerosol spray for enhancing/refracting the signalling of radio/radar waves along military jet flight paths, missile test ranges, etc. It is proposed that chronic contamination of the biosystem with the reactive types of Ba salts can initiate the pathogenesis of MS; due to the conjugation of Ba with free sulphate, which subsequently deprives the endogenous sulphated proteoglycan molecules (heparan sulfates) of their sulphate co partner, thereby disrupting synthesis of S-proteoglycans and their crucial role in the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signalling which induces oligodendrocyte progenitors to maintain the <em>growth</em> and structural integrity of the myelin sheath. Loss of S-proteoglycan activity explains other key facets of MS pathogenesis; such as the aggregation of platelets and the proliferation of superoxide generated oxidative stress. Ba intoxications disturb the sodium-potassium ion pump--another key feature of the MS profile. The co-clustering of various neurodegenerative diseases in these Ba-contaminated ecosystems suggests that the pathogenesis of all of these diseases could pivot upon a common disruption of the sulphated proteoglycan-<em>growth</em> <em>factor</em> mediated signalling systems. Individual genetics dictates which specific disease emerges at the end of the day.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are a family of <em>growth</em> <em>factors</em> critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs). Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and <em>growth</em>. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/<em>19</em> and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.