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

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of 22 members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-<em>17</em> target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process <em>growth</em> via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.

BACKGROUND

Vascular calcifications (VCs) are frequently observed in chronic kidney disease (CKD) and haemodialysis (HD) patients. They have been associated with numerous factors, particularly hyperphosphataemia, excess calcium load, hypertension and increased mortality rate. The purpose of this study is to measure VCs in long-HD patients with good blood pressure and phosphate control, with the occasional use of sevelamer, using a plain radiological score to identify the associated factors and effects on the 1-year survival rate.

METHODS

We studied HD patients from one centre using a semi-quantitative score ranging from 0 to 3 according to the severity and extent of VCs. The following patients' characteristics were compared according to their VC scores: medical history, treatments, blood pressure, standard biological data, fibroblast growth factor (FGF) 23, osteoprotegerin (OPG), whole PTH, beta-crosslaps, bone alkaline phosphatases and bone mineral density scores. One-year survival analyses were also performed.

RESULTS

Among the 250 HD patients of the centre, 161 were studied; the mean age was 67.2 +/- 13 years, 45% of the subjects were females, 35% were diabetics, and they had been on dialysis for between 1-486 months (median: 45 months) with a 3 x 5-3 x 8 h dialysis schedule using 1.5 mmol/l dialysate calcium and providing a mean 2.25 +/- 0.5 Kt/V. Only 17% of the patients were free from VCs and 11% had severe VCs. The factors associated with VCs were classified into 'classic' (age, diabetes, male gender, tobacco use, inflammation, more frequent warfarin treatment and peripheral vascular and cardiac diseases) and 'non-traditional' (higher FGF-23 and OPG serum levels, low albumin serum levels and low alfacalcidol and CaCO(3) use). In logistic regression, only age, diabetes and FGF-23 serum levels were associated with VC scores of 2 and 3. The patients with a score of 3 had a higher 1-year mortality rate (RR 2.1; P = 0.01) as compared to patients with a 0 score.

CONCLUSIONS

A plain radiological score showed the high prevalence (83%) of VCs in HD patients in spite of a long and intensive dialysis strategy and adherence to guidelines. The main associated factors were classic factors such as ageing and diabetes. No relationship was found with blood pressure and phosphataemia that remained well controlled in long dialysis; the association with FGF-23 serum levels may aggregate some non-traditional risk factors. The harmful effects of VCs on survival require their systematic assessment and optimization of the potentially modifiable associated factors in CKD and HD patients.

OBJECTIVE

To examine and quantify neuroprotective and neurite-promoting activity on retinal ganglion cells (RGCs) after injury of the lens.

METHODS

In adult albino rats, penetrating lens injury was performed by intraocular injection. To test for injury-induced neuroprotective effects in vivo, fluorescence-prelabeled RGCs were axotomized by subsequent crush of the optic nerve (ON) with concomitant lens injury to cause cataract. The numbers of surviving RGCs were determined in retinal wholemounts and compared between the different experimental and control groups. To examine axonal regeneration in vivo, the ON was cut and replaced with an autologous piece of sciatic nerve (SN). Retinal ganglion cells with axons that had regenerated within the SN under lens injury or control conditions were retrogradely labeled with a fluorescent dye and counted on retinal wholemounts. Neurite regeneration was also studied in adult retinal explants obtained either after lens injury or without injury. The numbers of axons were determined after 1 and 2 days in culture. Putative neurotrophins (NTs) were studied within immunohistochemistry and Western blot analysis.

RESULTS

Cataractogenic lens injury performed at the same time as ON crush resulted in highly significant rescue of 746 +/- 126 RGCs/mm(2) (mean +/- SD; approximately 39% of total RGCs) 14 days after injury compared with controls without injury or with injection of buffer into the vitreous body (30 +/- 18 RGCs/mm(2)). When lens injury was performed with a delay of 3 days after ON crush, 49% of RGCs survived, whereas delay of 5 days still rescued 45% of all RGCs. In the grafting paradigm virtually all surviving RGCs after lens injury appeared to have regenerated an axon within the SN graft (763 +/- 114 RGCs/mm(2) versus 79 +/- <em>17</em> RGCs/mm(2) in controls). This rate of regeneration corresponds to approximately 40% of all RGCs. In the regeneration paradigm in vitro preceding lens injury and ON crush 5 days previous resulted in a maximum of regeneration of 273 +/- 39 fibers/explant after 1 day and 574 +/- 38 fibers/explant after 2 days in vitro. In comparison, in control retinal pieces without lens injury 28 +/- 13 fibers/explant grew out at 1 day, and 97 +/- 37 fibers/explant grew out at 2 days in culture. Immunohistochemical and Western blot analysis of potential NTs in the injured lens revealed no expression of ciliary neurotrophic <em>factor</em> (CNTF), brain-derived neurotrophic <em>factor</em> (BDNF), NT-4, nerve <em>growth</em> <em>factor</em> (NGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF).

CONCLUSIONS

The findings indicate that the lens contains high neuroprotective and neuritogenic activity, which is not caused by NT. Compared with the data available in the literature, this neuroprotection is quantitatively among the highest ever reported within the adult rat visual system.

Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures (neutrophil extracellular traps - NETs) have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis-related agents, such as cigarette smoke, magnesium silicate, bleomycin, or with generic NET inducers, such as phorbol 12-myristate 13-acetate, induced activation of lung <em>fibroblasts</em> (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL-<em>17</em> (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue <em>growth</em> <em>factor</em> expression, collagen production, and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET-derived components in LF differentiation and function. Furthermore, IL-<em>17</em> was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL-<em>17</em>-driven fibrosis. Additionally, autophagy was identified as the orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha-smooth muscle actin (α-SMA)-expressing <em>fibroblasts</em> in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together, these data suggest that both autophagy and NETs are involved not only in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases.

The <em>growth</em> autonomy of human tumor cells is considered due to the endogenous production of <em>growth</em> <em>factors</em>. Transcriptional expression of candidates for autocrine stimulatory <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), acidic FGF, and transforming <em>growth</em> <em>factor</em> type beta were determined in human brain tumors. Basic FGF was expressed abundantly in <em>17</em> of 18 gliomas, 20 of 22 meninglomas, and 0 of 5 metastatic brain tumors. The level of mRNA expression of acidic FGF in gliomas was significant. In contrast, transforming <em>growth</em> <em>factor</em> type beta 1 was expressed in all the samples investigated. The mRNA for basic FGF and its peptide were localized in tumor cells in vivo by in situ hybridization and immunohistochemistry, showing that basic FGF is actually produced in tumor cells. Our results suggest that tumor-derived basic FGF is involved in the progression of gliomas and meningiomas in vivo, whereas acidic FGF is expressed in a tumor origin-specific manner, suggesting that acidic FGF works in tandem with basic FGF in glioma tumorigenesis.

OBJECTIVE

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

METHODS

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

RESULTS

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

CONCLUSIONS

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

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

OBJECTIVE

The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA(1), in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA(1)-antagonist (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966).

METHODS

The potency and selectivity of AM966 for LPA(1) receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model.

RESULTS

AM966 was a potent antagonist of LPA(1) receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC(50)= <em>17</em> nM) from Chinese hamster ovary cells stably expressing human LPA(1) receptors and inhibited LPA-induced chemotaxis (IC(50)= 181 nM) of human IMR-90 lung <em>fibroblasts</em> expressing LPA(1) receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming <em>growth</em> <em>factor</em> beta1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid.

CONCLUSIONS

These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA(1) receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.

In our study, we present experimental evidence suggesting that curcumin exerts multiple different suppressive effects on human breast carcinoma cells in vitro. Our experiments demonstrate that curcumin's antiproliferative effects are estrogen dependent in ER (estrogen receptor)-positive MCF-7 cells, being more pronounced in estrogen-containing media and in the presence of exogenous <em>17</em>-beta estradiol. Curcumin inhibits the expression of ER downstream genes including pS2 and TGF-beta (transforming <em>growth</em> <em>factor</em>) in ER-positive MCF-7 cells, and this inhibition is also dependent on the presence of estrogen. Curcumin also decreases ERE (estrogen responsive element)-CAT activities induced by <em>17</em>-beta estradiol. In addition, we demonstrate that curcumin exerts strong anti-invasive effects in vitro that are not estrogen dependent in the ER-negative MDA-MB-231 breast cancer cells. These anti-invasive effects appear to be mediated through the downregulation of MMP-2 (matrix metalloproteinase) and the upregulation of TIMP-1 (tissue inhibitor of metalloproteinase), 2 common effector molecules that have been implicated in regulating tumor cell invasion. Our study also demonstrates that curcumin inhibits the transcript levels of 2 major angiogenesis <em>factors</em> VEGF (vascular endothelial <em>growth</em> <em>factor</em>) and b-FGF (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) mainly in ER-negative MDA-MB-231 cells.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> family of secreted signaling molecules is essential for patterning in the central nervous system. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>17</em> (Fgf<em>17</em>) has been shown to contribute to regionalization of the rodent frontal cortex. To determine how Fgf<em>17</em> signaling modulates behavior, both during development and in adulthood, we studied mice lacking one or two copies of the Fgf<em>17</em> gene. Fgf<em>17</em>-deficient mice showed no abnormalities in overall physical <em>growth</em>, activity level, exploration, anxiety-like behaviors, motor co-ordination, motor learning, acoustic startle, prepulse inhibition, feeding, fear conditioning, aggression and ol<em>factor</em>y exploration. However, they displayed striking deficits in several behaviors involving specific social interactions. Fgf<em>17</em>-deficient pups vocalized less than wild-type controls when separated from their mother and siblings. Elimination of Fgf<em>17</em> also decreased the interaction of adult males with a novel ovariectomized female in a social recognition test and reduced the amount of time opposite-sex pairs spent engaged in prolonged, affiliative interactions during exploration of a novel environment. After social exploration of a novel environment, Fgf<em>17</em>-deficient mice showed less activation of the immediate-early gene Fos in the frontal cortex than wild-type controls. Our findings show that Fgf<em>17</em> is required for several complex social behaviors and suggest that disturbances in Fgf<em>17</em> signaling may contribute to neuropsychiatric diseases that affect such behaviors.

Defects in heart development are the most common congenital abnormalities in humans, providing a strong incentive to learn more about the underlying causes. Previous studies have implicated the metalloprotease-disintegrins ADAMs (a disintegrin and metalloprotease) <em>17</em> and 19 as well as heparin binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) and neuregulins in heart development in mice. Here, we show that mice lacking both ADAMs <em>17</em> and 19 have exacerbated defects in heart development compared to mice lacking either ADAM, providing the first evidence for redundant or compensatory functions of ADAMs in development. Moreover, we identified additional compensatory or redundant roles of ADAMs 9 and 19 in morphogenesis of the mitral valve and cardiac outflow tract. Cell biological studies designed to address the functions of these ADAMs in shedding of HB-EGF uncovered a contribution of ADAM19 to this process, but this was only evident in the absence of the major HB-EGF sheddase, ADAM<em>17</em>. In addition, ADAM<em>17</em> emerged as the major sheddase for neuregulins beta1 and beta2 in mouse embryonic <em>fibroblasts</em>. These results raise the possibility that ADAMs 9, <em>17</em>, and 19 contribute to heart development in humans and have implications for understanding the mechanisms underlying congenital heart disease.

Purpose No standard treatment exists for patients with cholangiocarcinoma for whom first-line gemcitabine-based therapy fails. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 ( FGFR2) fusions/translocations are present in 13% to <em>17</em>% of intrahepatic cholangiocarcinomas. BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor, has shown preliminary clinical activity against tumors with FGFR alterations. Methods A multicenter, open-label, phase II study ( ClinicalTrials.gov identifier: NCT02150967) evaluated BGJ398 antitumor activity in patients age ≥ 18 years with advanced or metastatic cholangiocarcinoma containing FGFR2 fusions or other FGFR alterations whose disease had progressed while receiving prior therapy. Patients received BGJ398 125 mg once daily for 21 days, then 7 days off (28-day cycles). The primary end point was investigator-assessed overall response rate. Results Sixty-one patients (35 women; median age, 57 years) with FGFR2 fusion (n = 48), mutation (n = 8), or amplification (n = 3) participated. At the prespecified data cutoff (June 30, 2016), 50 patients had discontinued treatment. All responsive tumors contained FGFR2 fusions. The overall response rate was 14.8% (18.8% FGFR2 fusions only), disease control rate was 75.4% (83.3% FGFR2 fusions only), and estimated median progression-free survival was 5.8 months (95% CI, 4.3 to 7.6 months). Adverse events included hyperphosphatemia (72.1% all grade), fatigue (36.1%), stomatitis (29.5%), and alopecia (26.2%). Grade 3 or 4 treatment-related adverse events occurred in 25 patients (41%) and included hyperphosphatemia (16.4%), stomatitis (6.6%), and palmar-plantar erythrodysesthesia (4.9%). Conclusion BGJ398 is a first-in-class FGFR kinase inhibitor with manageable toxicities that shows meaningful clinical activity against chemotherapy-refractory cholangiocarcinoma containing FGFR2 fusions. This promising antitumor activity supports continued development of BGJ398 in this highly selected patient population.

The <em>fibroblast</em> <em>growth</em> <em>factor</em>-related protooncogenes, int-2 and hst/k-FGF, are within <em>17</em> kilobase pairs of one another on mouse chromosome 7 and are in the same transcriptional orientation. Approximately 70% of tumors induced in BR6 mice by mouse mammary tumor virus have proviral insertions adjacent to the int-2 gene. We find that the murine homolog of the hst/k-FGF gene can also be transcriptionally activated by the insertion of mouse mammary tumor virus DNA either upstream or downstream of the gene. In most tumors, only one of these adjacent genes is activated, but in some cases both genes are expressed. One of the hst-expressing tumors also has a virally activated int-3 gene. At least five distinct cellular genes (int-1, -2, -3, -4, and hst/k-FGF) can therefore contribute, either singly or in concert, to the development of histologically indistinguishable mammary tumors in mice infected by mouse mammary tumor virus.

It has been suggested that IL-<em>17</em>RC forms a complex with IL-<em>17</em>RA to mediate the functions of IL-<em>17</em>A and IL-<em>17</em>F homodimers as well as IL-<em>17</em>A/F heterodimers. It is still unclear whether IL-<em>17</em>RC is absolutely required for the signaling of IL-<em>17</em> cytokines in vivo. By using Il-<em>17</em>rc-deficient mice, we show that IL-<em>17</em>RC is essential for the signaling of IL-<em>17</em>A, IL-<em>17</em>F, and IL-<em>17</em>A/F both in vitro and in vivo. IL-<em>17</em>RC does not preassociate with IL-<em>17</em>RA on the cell surface; rather IL-<em>17</em>A can induce the formation of an IL-<em>17</em>RC and IL-<em>17</em>RA complex. This process is not dependent on the intracellular similar expression to <em>fibroblast</em> <em>growth</em> <em>factor</em> genes and IL-<em>17</em>Rs (SEFIR) domain of IL-<em>17</em>RC, but the SEFIR is essential in IL-<em>17</em>A signal transduction. Finally, Il-<em>17</em>rc(-/-) mice develop much milder disease in an experimental autoimmune encephalomyelitis model, supporting an essential role for IL-<em>17</em>RC in mediating immune-mediated CNS inflammation.

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non-small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERalpha, ERbeta, and ER coregulators in human lung <em>fibroblasts</em> and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERbeta and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERalpha or the p160 coactivator SRC-1. ERbeta and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist <em>17</em>-beta-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal <em>growth</em> <em>factor</em>, which served as a positive control for an alternative <em>growth</em> stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal <em>growth</em> <em>factor</em> also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

BACKGROUND

Fetal skin wound healing results in scarless repair with minimal cellular inflammatory response. Interleukin-8 (IL-8) stimulates inflammation in postnatal wound healing but little is known about its role in fetal wounds. We hypothesized that fetal tissues have diminished IL-8 during wound repair and in response to platelet-derived growth factor (PDGF), a growth factor central to wound healing.

METHODS

To examine the IL-8 response of fibroblasts to PDGF, cultures of human fetal (17-18 weeks) and adult dermal fibroblasts were incubated 8 h with PDGF (0, 0.1, 1, or 10 ng/mL) and supernatants and cells were collected for IL-8 ELISA and IL-8 RT-PCR. To evaluate the IL-8 response to wounding, human adult and fetal skin was placed subcutaneously in the SCID mouse, wounded, and the wound cleft excised after 4, 12, 24, or 72 h for IL-8 RT-PCR.

RESULTS

Fetal fibroblasts produced less IL-8 protein at baseline (50 +/- 6 pg/mL versus 450 +/- 115 pg/mL, P < 0.001) and in response to all concentrations of PDGF examined (P < 0.001). IL-8 mRNA was detected in unstimulated adult fibroblasts but not in fetal fibroblasts. Much less IL-8 mRNA was detected in stimulated fetal fibroblasts than in adult fibroblasts. IL-8 mRNA was detected 4 h after wounding in fetal and adult wounds. By 12 h no IL-8 mRNA was detected in fetal wounds, whereas adult wounds had IL-8 mRNA persisting to 72 h.

CONCLUSIONS

Diminished inflammatory cytokine response by fetal tissues may be responsible for the lack of cellular recruitment and inflammation seen in fetal wound healing and may contribute to scarless wound repair.

The zeta isoform of protein kinase C (zeta PKC) has been shown to be involved in the maturation of Xenopus oocytes and mitogenic signaling in <em>fibroblasts</em>. zeta PKC also regulates the important transcription <em>factor</em> nuclear <em>factor</em> kappa B, most probably by phosphorylation of the inhibitory molecule I kappa B. The mechanisms that control zeta PKC activity are still poorly characterized. This kinase is not activated by diacylglycerol but is potently stimulated in vitro by the products of phosphatidylinositol 3-kinase (PI 3-kinase), which suggests that zeta PKC is at least one of the critical targets of PI 3-kinase-triggered signals, and strengthens its role in cell proliferation. PI 3-kinase has been shown, like Raf, to be a direct effector of Ras. zeta PKC is a required step for Ras mitogenic signaling. Therefore, it is possible that zeta PKC directly interacts with Ras during mitogenic activation. We demonstrate here that Ras interacts in vitro with the regulatory domain of zeta PKC as well as that the association of zeta PKC with Ras in vivo is triggered by platelet-derived <em>growth</em> <em>factor</em>. It is also shown here that the expression of a dominant negative mutant of Ras (Asn-<em>17</em>) severely impairs the activation of zeta PKC in mouse <em>fibroblasts</em>.

To examine which <em>growth</em> <em>factors</em> correlate with neovascularization in human brain tumors, the mRNA levels of transforming <em>growth</em> <em>factor</em> alpha, transforming <em>growth</em> <em>factor</em> beta, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand <em>factor</em>. We normalized the <em>growth</em> <em>factor</em> mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the <em>17</em> gliomas and 16 meningiomas examined, the mRNA of transforming <em>growth</em> <em>factors</em> alpha and beta, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and VEGF were expressed at various levels. Among those 4 <em>growth</em> <em>factors</em>, the mRNA levels of VEGF, but not those of transforming <em>growth</em> <em>factors</em> alpha and beta and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive <em>factor</em> in tumor angiogenesis in both human gliomas and meningiomas.

We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for <em>growth</em> in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, <em>17</em> alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.

Oncogenic signaling through activation of epidermal <em>growth</em> <em>factor</em> receptor (EGFR), HER-2, and hypoxia inducible-<em>factor</em>-1alpha (HIF-1alpha) has been implicated in gastric cancer <em>growth</em> and angiogenesis through up-regulation of vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1alpha. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor <em>growth</em>. In vitro, the geldanamycin derivate <em>17</em>-allylamino-<em>17</em>-demethoxygeldanamycin (<em>17</em>-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1alpha protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, <em>17</em>-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by <em>17</em>-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of <em>17</em>-AAG in vitro were higher on cancer cells and gastric <em>fibroblasts</em> than on pericytes. In vivo, the water-soluble compound <em>17</em>-dimethylaminoethylamino-<em>17</em>-demethoxygeldanamycin (<em>17</em>-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor <em>growth</em>. By immunohistochemistry, <em>17</em>-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant <em>growth</em>-inhibitory effects of <em>17</em>-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor <em>growth</em> in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens.

Cross-talk between G protein-coupled receptor (GPCR) and epidermal <em>growth</em> <em>factor</em> receptor (EGFR) signaling systems is widely established in a variety of normal and transformed cell types. Here, we demonstrate that the EGFR transactivation signal requires metalloproteinase cleavage of epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em> precursors in <em>fibroblasts</em>, ACHN kidney, and TccSup bladder carcinoma cells. Furthermore, we present evidence that blockade of the metalloproteinase-disintegrin tumor necrosis <em>factor</em>-alpha-converting enzyme (TACE/ADAM<em>17</em>) by a dominant negative ADAM<em>17</em> mutant prevents angiotensin II-stimulated pro-HB-EGF cleavage, EGFR activation, and cell proliferation in ACHN tumor cells. Moreover, we found that in TccSup cancer cells, the lysophosphatidic acid-induced transactivation signal is mediated by ADAM15, demonstrating that distinct combinations of <em>growth</em> <em>factor</em> precursors and ADAMs (a disintegrin and metalloproteinases) regulate GPCR-EGFR cross-talk pathways in cell lines derived from urogenital cancer. Our data show further that activation of ADAMs results in discrete cellular responses; whereas GPCR agonists promote activation of the Ras/MAPK pathway and cell proliferation via the EGFR in <em>fibroblasts</em> and ACHN cells, EGFR transactivation pathways regulate activation of the survival mediator Akt/protein kinase B and the susceptibility of <em>fibroblasts</em> and TccSup bladder carcinoma cells to proapoptotic signals such as serum deprivation, death receptor stimulation, and the chemotherapeutic drug doxorubicin. Thus, ADAM15 and -<em>17</em> function as effectors of GPCR-mediated signaling and define critical characteristics of cancer cells.

An improved Ham's F12 nutrient medium supplemented with epidermal <em>growth</em> <em>factor</em> (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal <em>growth</em> of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measureable stimulation on TE cell <em>growth</em> and plating efficiency. However, serum levels higher than 0.1% inhibited cell <em>growth</em> and also masked the <em>growth</em> stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal <em>growth</em> occurred at a seeding density of <em>17</em> cells/cm2 with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a <em>fibroblast</em> feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) is a hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism. Recent observational studies in humans suggest that circulating FGF23 is independently associated with cardiac hypertrophy and increased mortality, but it is unknown whether FGF23 can directly alter cardiac function. We found that FGF23 significantly increased cardiomyocyte cell size in vitro, the expression of gene markers of cardiac hypertrophy, and total protein content of cardiac muscle. In addition, FGFR1 and FGFR3 mRNA were the most abundantly expressed FGF receptors in cardiomyocytes, and the coreceptor α-klotho was expressed at very low levels. We tested an animal model of chronic kidney disease (Col4a3(-/-) mice) that has elevated serum FGF23. We found elevations in common hypertrophy gene markers in Col4a3(-/-) hearts compared with wild type but did not observe changes in wall thickness or cell size by week 10. However, the Col4a3(-/-) hearts did show reduced fractional shortening (-<em>17</em>%) and ejection fraction (-11%). Acute exposure of primary cardiomyocytes to FGF23 resulted in elevated intracellular Ca(2+) ([Ca(2+)](i); F/F(o) + 86%) which was blocked by verapamil pretreatment. FGF23 also increased ventricular muscle strip contractility (67%), which was inhibited by FGF receptor antagonism. We hypothesize that although FGF23 can acutely increase [Ca(2+)](i), chronically this may lead to decreases in contractile function or stimulate cardiac hypertrophy, as observed with other stress hormones. In conclusion, FGF23 is a novel bone/heart endocrine <em>factor</em> and may be an important mediator of cardiac Ca(2+) regulation and contractile function during chronic kidney disease.