BACKGROUND

Aberrant wound healing of skin injury may lead to 2 pathologic entities, termed keloids and hypertrophic scars (HS). There has been growing evidence suggesting a role for transforming growth factor beta (TGF-β) family members in the pathogenesis of fibrosis.

OBJECTIVE

The aim of the present work was to investigate the role of TGF-β1 in the pathogenesis of keloids and HS.

METHODS

TGF-β1 was analyzed on skin biopsies of 30 patients presenting with keloids (16) or HS (14) and 10 normal surgical scar and 10 age- and sex-matched normal subjects (controls).

RESULTS

TGF-β1 was expressed in dermal fibroblasts, inflammatory cells, and endothelial cells of normal surgical scar (60%) and aberrant scar (86.7%) with an absence of statistical difference. Although it is expressed in 90% of epidermis of aberrant scar (diffuse expression) compared with 60% of normal surgical scar (basal layer expression) and 20% of normal skin biopsies (basal layer expression) with highly significant differences. Dermal TGF-β1 expression in aberrant scar lesions was significantly associated with lesions of shorter duration (P = 0.01) and older age group (P = 0.02). The intense dermal expression was also associated with lesions of shorter duration (P = 0.0001) and immature scars (P = 0.002).

CONCLUSIONS

TGF-β1 is involved in the pathogenesis of both keloids and HS with maximum effect at early stages. Keratinocytes are not passive partners but rather may have an active role in the induction of fibrosis. Targeting of TGF-β1 may be of benefit if applied early and if directed against keratinocytes as an unusual target of therapy.

Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the beta(1) integrin and the <em>16</em>-kDa subunit of vacuolar H(+)-ATPase (<em>16</em>K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged <em>16</em>K demonstrate co-localization with beta(1) integrin in focal adhesions. Deletion of the fourth of four transmembrane helices in <em>16</em>K results in loss of interaction with beta(1) integrin in vitro and in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of platelet-derived <em>growth</em> <em>factor</em>-beta receptor signaling by the human papillomavirus E5 oncoprotein. Overexpression of <em>16</em>K or expression of <em>16</em>K lacking this helix alters the morphology of myoblasts and <em>fibroblasts</em>, suggesting that the interaction of <em>16</em>K with integrins could be important for cell <em>growth</em> control. We also discuss the possible role <em>16</em>K might play in integrin movement.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, <em>16</em>, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/<em>16</em>/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, <em>16</em>, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.

Skin <em>fibroblast</em> cultures were established from mouse foetuses at days 14, 15, <em>16</em> and 18 of gestation and from newborn mice. Modulations of mitotic and biosynthetic phenotypes of the <em>fibroblasts</em> by transforming <em>growth</em> <em>factor</em> beta1 (TGFbeta1) were studied. Treatment of the <em>fibroblasts</em> with TGFbeta1 at doses of 1 and 10 ng/ml for 48 h resulted in significant stimulation of cell proliferation in the 15-, <em>16</em>- and 18-day foetal <em>fibroblasts</em> and a slight stimulation in the 14-day foetal <em>fibroblasts</em>. Treatment with TGFbeta1 resulted in stimulation of collagen synthesis approximately 2-fold in the 18-day foetal and newborn <em>fibroblasts</em>, but failed to stimulate it in the 14-, 15- and <em>16</em>-day foetal <em>fibroblasts</em>. TGFbeta1 stimulated glycosaminoglycan (GAG) synthesis throughout all developmental stages approximately 1.8-2.6 fold. Histological study demonstrated that skin wounds made at day <em>16</em> of gestation were replaced with normal-appearing dermis, but at day 18 the wounds left dermal fibrosis and lack of hair follicles. These results indicate that the modulations of <em>fibroblast</em> phenotypes (proliferation and syntheses of collagen and GAG) in response to TGFbeta1 occur at different stages of gestation. Ontogenic transitions of skin wound healing and collagen synthetic phenotype with TGFbeta1 treatment in cultured <em>fibroblasts</em> occurred between days <em>16</em> and 18 of gestation, suggesting that the unresponsiveness of collagen synthesis to exogenous TGFbeta1 in cell culture may be related to the phenomenon of scarless wounds in the foetus.

Interleukin 6 (IL-6) is a potent cytokine, the biological activities of which include the stimulation of immunoglobulin secretion, T cell activation, induction of the acute phase response, activation of megakaryocytes, and pyrogenicity. These biological activities make it a plausible contributor to rheumatoid arthritis. The ability of synoviocytes to synthesise this potential mediator of inflammation was tested. Cultures of <em>fibroblast</em>-like cells were established from joint tissue from patients with rheumatoid arthritis, degenerative joint disease, or trauma. Supernatants from synoviocytes from each diagnostic category contained IL-6-like activity as detected in a B9 plasmacytoma cell proliferation assay. Supernatants from IL-1 stimulated synoviocytes from patients with rheumatoid arthritis (n = 5) contained an average of 70,000 U/ml IL-6. Western blot analysis confirmed that these supernatants contained peptides that reacted with a highly specific antibody to IL-6. A cDNA probe specific for IL-6 hybridised with mRNA derived from synoviocytes representative of each disease state. Interleukin 6 mRNA expression increased by culturing synoviocytes in the presence of 10% calf serum, IL-1 (30 U/ml), insulin (<em>16</em>6 ng/ml), or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (<em>16</em> ng/ml). In contrast, dexamethasone (10(-6) mol/l) suppressed the ability of IL-1 to increase the expression of IL-6 mRNA. Recombinant IL-6 itself did not detectably upregulate its own message. The regulation of production of IL-6 by synoviocytes may be important in the pathogenesis of joint inflammation.

We analyzed the expression of mRNAs for <em>growth</em> <em>factor</em> [epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and platelet-derived <em>growth</em> <em>factor</em> A chain (PDGF-A)] and their receptor (R) by reverse transcription-polymerase chain reaction in bovine ova during oocyte maturation in vitro (0-21 hr) and after fertilization in vitro (6-144 hr: zygotes to blastocysts). Transcripts for EGF were not found before fertilization. Transcripts for IGF-I were present in immature oocytes immediately after collection and in embryos from the 2-cell stage onward. Transcripts for bFGF were present in all stages of oocyte maturation and after fertilization up to the <em>16</em>-cell stage. Transcripts for PDGF-A were present in all stages of oocyte maturation and after fertilization up to the 2-cell stage. Transcripts for ErbB3 (a member of the EGF-R subfamily), and bFGF-R were present in all stages of oocyte maturation, after fertilization up to the 2-cell stage, and the blastocyst stage. Transcripts for IGF-I-R and PDGF-Ralpha were present in all stages of oocyte maturation and embryo development. The results of this study showed that eight different messages for <em>growth</em> <em>factor</em> and their receptor were detectable in bovine ova during oocyte maturation and/or after fertilization in vitro and their expression patterns were the gene-specific rather than the developmental stage of bovine ova.

BACKGROUND

Human papillomavirus type <em>16</em> (HPV-<em>16</em>) E5 protein co-operates with epidermal <em>growth</em> <em>factor</em> to stimulate mitogenesis of murine <em>fibroblasts</em>. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-<em>16</em> E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-<em>growth</em> of murine <em>fibroblasts</em>.

RESULTS

We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-<em>16</em> abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-<em>16</em> E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-<em>growth</em>; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-<em>growth</em> and repression of p21 expression, despite high transcription levels.

CONCLUSIONS

The effects of HPV-<em>16</em> E5 variants upon mitosis help to explain why Leu44Val65 HPV-<em>16</em> E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK.

OBJECTIVE

Modulating cytokine signaling in vocal fold fibroblasts after injury may influence extracellular matrix (ECM) production and eventual fibrotic outcome. To evaluate previously established in vivo cytokine and ECM gene expression hypotheses, we examined in vitro vocal fold fibroblast responses to exogenous inflammatory factor stimulation.

METHODS

Rat vocal fold fibroblast lines derived from explants were separately treated with interleukin-13 (IL-13), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta subtype 1 (TGF-beta1), or prostaglandin E2 (PGE2). We examined the in vitro messenger RNA expression profiles of IL-1beta, IFN-gamma, TNF-alpha, TGF-beta1, and cyclooxygenase 2 (COX-2), as well as those of hyaluronic acid synthase (HAS) 1, HAS-2, procollagen subtype 1, and procollagen subtype 3, at 1,4, 8, 16, 24, and 72 hours after treatment, and compared them to those of untreated fibroblasts and in vivo data, using real-time reverse transcription-polymerase chain reaction.

RESULTS

IL-1beta and TNF-alpha induced each other and synergistically increased HAS-1 and HAS-2 expression. PGE2 also up-regulated HAS-1 and HAS-2 expression. IFN-gamma, IL-1beta, TNF-alpha, and TGF-beta1 up-regulated HAS expression alongside either transient up-regulation of, or no change in, procollagen 1 and 3 expression. Most treatments appeared to suppress procollagen expression, possibly through HAS up-regulation. All inflammatory factors attenuated TGF-beta1 expression.

CONCLUSIONS

These results confirm several in vivo trends, identify potential cytokine pathways and therapeutic candidates, and suggest the utility of this in vitro setup for future studies.

GH is one of the major <em>factors</em> required for the differentiation of 3T3-F442A preadipocyte <em>fibroblasts</em> into adipocytes. An early event following the addition of GH to 3T3-F442A preadipocytes is induction of the expression of c-fos and c-jun. Although c-fos and c-jun expression has been observed in conjunction with <em>growth</em> <em>factor</em>-stimulated differentiation in several cell types, it is not clear whether protooncogene expression and differentiation are necessarily related. In this study the relationship between the induction of these protooncogenes and differentiation was evaluated by taking advantage of several cell lines that are related to 3T3-F442A cells but have varying GH requirements for differentiation. Adipose differentiation in the adipogenic cell lines 3T3-L1 and 3T3-GI-<em>16</em> is known to be GH independent, requiring insulin or insulin-like <em>growth</em> <em>factor</em>-I. In both 3T3-L1 and 3T3-GI-<em>16</em> preadipocytes, GH, nevertheless, induced the expression of mRNA for both protooncogenes. GH (2.2 nM) was more effective than insulin (1 microM) in inducing c-fos expression in these two adipogenic cell lines, suggesting that induction of the protooncogenes is not sufficient to induce adipogenesis. 3T3-C2 <em>fibroblasts</em> do not differentiate in response to any of the stimuli that convert 3T3-F442A <em>fibroblasts</em> to adipocytes. However, GH (2.2 nM) as well as calf serum induced the expression of c-fos and c-jun in 3T3-C2 cells. NIH-3T3 <em>fibroblasts</em>, which do not undergo differentiation, also showed induction of c-fos by GH. Thus, GH-induced expression of c-fos and c-jun occurs in nondifferentiating cells. Furthermore, in differentiated 3T3-F442A adipocytes, GH stimulated the expression of c-fos and c-jun as it does in the preadipocytes. Since GH elicits a variety of metabolic responses in 3T3-F442A adipocytes, the present findings raise the possibility that induction of c-fos and c-jun expression might be associated with multiple events in GH-stimulated 3T3-F442A adipocytes. The lack of requirement for GH in GH-independent and nondifferentiating cells compared to 3T3-F442A cells does not appear to reflect the lack of GH receptors, since expression of mRNA for the GH receptor was evident in all of the cell types tested and, thus, corresponds with the ability of GH to induce protooncogene expression. Although GH-induced c-fos expression was relatively invariant, since it was evident in all of the cell types studied, this response could clearly be regulated, since it was attenuated by prior exposure to GH or serum in 3T3-F442A preadipocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

For various forms of human glomerulonephritis a close relationship between inflammatory injury and a local mesangial proliferative response has been described. Herein, we used primary cultures of human glomerular mesangial cells (HMCs) from five different donors to determine the autocrine <em>growth</em>-inducing capacity of their supernatants after stimulation with different cytokines and lipopolysaccharide (LPS) to determine whether this effect is due to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The basal <em>growth</em>-inducing capacity of supernatants collected from serum-free cultured HMC and concentrated 100-fold above a cut-off size of 10 kd was significantly increased by interleukin (IL)-1 beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF), and LPS up to 15-fold, but not by IL-1 alpha, IL-6, or bFGF. An anti-human bFGF antibody blocked the majority of IL-1 or LPS-induced proliferative effect of supernatants; complete inhibition was achieved by a combination of anti-human bFGF- and anti-human platelet-derived <em>growth</em> <em>factor</em> antibodies. HMCs express different isoforms of bFGF (18, 21.5, and 24 kd) in membrane, cytosolic, and nuclear fractions. All isoforms of bFGF were found in the nuclear fraction of HMC, whether stimulated or not. Immunoblots for bFGF protein of HMC demonstrated that only a approximate to <em>16</em> kd bFGF protein was released into HMC supernatants after stimulation with IL-1 beta, platelet-derived <em>growth</em> <em>factor</em>-BB, and LPS. The 18 kd isoform of bFGF accumulated in the membranes but was not released after stimulation with IL-1 alpha, IL-6, and bFGF, suggesting that its release was a prerequisite for autocrine <em>growth</em> stimulation. By means of reverse transcription polymerase chain reaction controlled by Southern blots, bFGF-mRNA expression of HMC was enhanced by IL-1 alpha, IL-1 beta, and LPS. Finally, we were able to show that HMCs are expressing bFGF receptors. In summary, our data demonstrate for the first time that the autocrine proliferative response of HMC to major inflammatory <em>factors</em> may primarily be mediated by bFGF.

With increasing efforts to develop and utilize mouse models of a variety of neuro-developmental diseases, there is an urgent need for sensitive neuroimaging methods that enable in vivo analysis of subtle alterations in brain anatomy and function in mice. Previous studies have shown that the brains of <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 17 null mutants (Fgf17(-/-)) have anatomical abnormalities in the inferior colliculus (IC)-the auditory midbrain-and minor foliation defects in the cerebellum. In addition, changes in the expression domains of several cortical patterning genes were detected, without overt changes in forebrain morphology. Recently, it has also been reported that Fgf17(-/-) mutants have abnormal vocalization and social behaviors, phenotypes that could reflect molecular changes in the cortex and/or altered auditory processing / perception in these mice. We used manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) to analyze the anatomical phenotype of Fgf17(-/-) mutants in more detail than achieved previously, detecting changes in IC, cerebellum, olfactory bulb, hypothalamus and frontal cortex. We also used MEMRI to characterize sound-evoked activity patterns, demonstrating a significant reduction of the active IC volume in Fgf17(-/-) mice. Furthermore, tone-specific (<em>16</em>- and 40-kHz) activity patterns in the IC of Fgf17(-/-) mice were observed to be largely overlapping, in contrast to the normal pattern, separated along the dorsal-ventral axis. These results demonstrate that Fgf17 plays important roles in both the anatomical and functional development of the auditory midbrain, and show the utility of MEMRI for in vivo analyses of mutant mice with subtle brain defects.

Cigarette smoking is an important risk <em>factor</em> for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight [BW]) or saline for <em>16</em> weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1 mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for <em>16</em> weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P < 0.05) attenuated the HFD-induced decrease in <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) and silent information regulator 1 (SIRT1). We conclude that nicotine, when combined with HFD, triggers CM apoptosis through the generation of oxidative stress and inactivation of AMPK together with the activation of caspase-2-mediated intrinsic apoptotic signaling independently of FGF21 and SIRT1.

OBJECTIVE

To establish human corneal stroma- and sclera-derived cells as models for studying diseases of the anterior segment of the eye.

METHODS

Using a recombinant retrovirus system, we transfected human papilloma virus <em>16</em> E6 and E7 (HPV<em>16</em> E6/E7) into human corneal stroma- and sclera-derived cells. The primary cells and established cell strains were characterized by assessing the mRNA expression of collagen, matrix metalloproteinase, and tissue inhibitor of metalloproteinase by reverse transcription-polymerase chain reaction. We also examined the effects of inflammatory cytokines on hyaluronan synthase expression and hyaluronan products.

RESULTS

Both a corneal stroma-derived cell strain, Cs3, and a sclera-derived cell strain, Sc1, were obtained, and both cell strains could be passaged up to 25 times. The mRNA expression pattern observed in the primary cells was identical to that observed in the cell strains. Hyaluronan synthase 1 and 2 mRNAs were increased by transforming growth factor beta and platelet-derived growth factor BB. Significant differences were observed between the hyaluronan products with and without cytokine treatment.

CONCLUSIONS

Cell strains derived from corneal stroma and sclera fibroblast cells can be established using HPV<em>16</em> E6/E7 immortalized genes of the same origin. The phenotypic cell characteristics did not change after transfection, immortalization, or successive passages in culture.

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the <em>16</em>-h assay period. Several hormones and <em>growth</em> <em>factors</em> were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and a partially purified preparation of sarcoma <em>growth</em> <em>factor</em>, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.

BACKGROUND

Transforming growth factor (TGF)-beta1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 receptors, and FGF-2 and its R1 receptor in murine dura cells.

METHODS

A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction.

RESULTS

Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4.

CONCLUSIONS

These results indicate that TGF-beta1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

Results from genome-wide association studies (GWAS) have indicated that strong single-gene effects are the exception, not the rule, for most diseases. We assessed the joint effects of germline genetic variations through a pathway-based approach that considers the tissue-specific contexts of GWAS findings. From GWAS meta-analyses of lung cancer (12 <em>16</em>0 cases/<em>16</em> 838 controls), breast cancer (15 748 cases/18 084 controls) and prostate cancer (14 <em>16</em>0 cases/12 724 controls) in individuals of European ancestry, we determined the tissue-specific interaction networks of proteins expressed from genes that are likely to be affected by disease-associated variants. Reactome pathways exhibiting enrichment of proteins from each network were compared across the cancers. Our results show that pathways associated with all three cancers tend to be broad cellular processes required for <em>growth</em> and survival. Significant examples include the nerve <em>growth</em> <em>factor</em> (P = 7.86 × 10(-33)), epidermal <em>growth</em> <em>factor</em> (P = 1.18 × 10(-31)) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (P = 2.47 × 10(-31)) signaling pathways. However, within these shared pathways, the genes that influence risk largely differ by cancer. Pathways found to be unique for a single cancer focus on more specific cellular functions, such as interleukin signaling in lung cancer (P = 1.69 × 10(-15)), apoptosis initiation by Bad in breast cancer (P = 3.14 × 10(-9)) and cellular responses to hypoxia in prostate cancer (P = 2.14 × 10(-9)). We present the largest comparative cross-cancer pathway analysis of GWAS to date. Our approach can also be applied to the study of inherited mechanisms underlying risk across multiple diseases in general.

We report the genomic structure and entire sequence of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4) gene. The gene spans approximately 11.3 kb. It is composed of 18 exons ranging in size from 71 bp to 600 bp. Exon-intron boundaries follow the GT/AG rule. Exon 1 is untranslated and preceded by structural elements characteristic of a TATA-free promoter. Although there are promoter motifs in intron 4 as well, there is currently no evidence of alternative transcription of FGFR4. Comparison of exon-intron boundaries of FGFR4 with those of FGFR1 and 3 reveals a remarkable degree of homology. With the exception of four, exon boundaries are at identical positions in all three receptor genes. Short tandem repeat polymorphisms (STRPs) were identified in introns 2 and <em>16</em> of FGFR4. The STRPs together with the sequence information will facilitate the rapid analysis of FGFR4 in those human disorders in which this gene can be considered a candidate.

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal <em>growth</em> <em>factor</em> (EGF), insulin, cholera toxin, and endothelial cell <em>growth</em> supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding <em>growth</em> <em>factors</em>, and in this report we have compared the effect of ECGS to purified heparin-binding <em>growth</em> <em>factors</em>. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding <em>growth</em> <em>factors</em>, alpha-endothelial cell <em>growth</em> <em>factor</em> (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGFa) stimulated [3H]thymidine incorporation by <em>16</em>-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding <em>growth</em> <em>factors</em> for both endothelial cells and <em>fibroblasts</em> but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding <em>growth</em> <em>factors</em> had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Benign prostatic hyperplasia (BPH) is a condition that affects a significant population of older males, yet its pathogenesis is not clearly understood. This study was designed to give broader insight into the early development of BPH by looking at changes in <em>growth</em> <em>factor</em> production in the ventral prostate. To accomplish this, Sprague Dawley rats (n = <em>16</em>, 250-300 g) were randomly divided into four equal groups. Three treatment groups were each implanted with ceramic drug delivery devices that were designed to deliver continuous physiologic doses of testosterone (T), dihydrotestosterone (DHT), and androstenedione (AED) for ninety-day duration. Immuno-histochemical analysis for epidermal <em>growth</em> <em>factor</em> (EGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) indicated whether these <em>growth</em> <em>factors</em> were involved in early processes of BPH induced by the delivery of androgens. Histopathological evaluation demonstrated increased positive reactivity for both EGF and bFGF in all steroid treated animals compared to controls. A similar trend was observed in the vascular endothelium. This information could be helpful in developing new methods for early diagnosis of BPH, but more importantly this knowledge provides the literature with clues about the cellular responses encountered at the initiation of the disease process.

<em>Fibroblast</em> cell population kinetics in the developing molar periodontal ligament was investigated in 10, 12, <em>16</em> and 20 days old mice by autoradiography after the administration of [3H]thymidine. Labelled mitoses, in number per unit area, were counted over the apical zones of the sections and percentage labelled mitoses (PLM) curves were drawn. Median values for some phases durations were read off at the 0.5 level of mitotic labelling. In determining other kinetic parameters the periodontal <em>fibroblast</em> population was considered separately as (1) a steady state system, (2) an exponentially <em>growing</em> system. An attempt was made to estimate mean values for these parameters using the Gilbert Computer programme. The programme generated the original data points and the fitted curve in graphical and numerical form together with the mean values and standard errors. The fact that the Gilbert programme assumes a stationary population in its theoretical PLM curve analysis was used to establish the kinetic type of the periodontal <em>fibroblast</em> population. The present study has demonstrated that in the <em>growing</em> periodontal ligament where cell specialisation and migration occurs steady state system is the kinetic model applicable. Failure of the computed PLM curves to fit the data adequately confirmed the <em>fibroblast</em> migration (apico-occlusal migration) from the apical zone to other zones. A definite cytokinetic basis for the periodontal ligament <em>fibroblast</em> proliferation and migration was established. Accordingly, <em>fibroblast</em> proliferation and migration takes place by a reduction in the DNA synthesis time (T8), cell generation tie (Tc) and possibly by a similar reduction in other parameters. Maximum reduction in these parameters is associated with the peak proliferative and migratory activity in the 12 days old group which is also the time that tooth eruption takes place in the mouse. Thus <em>fibroblast</em> proliferation and migration are major causative <em>factors</em> responsible for tooth eruption. Based on these results, a mechanism for tooth eruption is proposed.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>16</em> (FGF-<em>16</em>) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-<em>16</em> (rFGF-<em>16</em>) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-<em>16</em>, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-<em>16</em> extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-<em>16</em> induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-<em>16</em>, a novel FGF family member, has both unique structural and biological properties.

Titanium-aluminum-vanadium wear particles isolated from the soft-issue membrane of a failed total hip arthroplasty were added to human <em>fibroblasts</em> in cell culture. The cellular response to particle challenge was determined by assaying for levels of interleukin-1 beta, interleukin-6, tumor necrosis <em>factor</em>-alpha, prostaglandin E2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-AB, and transforming <em>growth</em> <em>factor</em>-beta. Collagenase and gelatinase activities were analyzed by zymography and [3H]collagen degradation. Cell viability was assessed by measuring the uptake of [3H]thymidine. Over the range of particle concentrations tested, cell viability, as demonstrated by [3H]thymidine uptake, remained unaffected. <em>Fibroblasts</em> exhibited a dose-dependent release of interleukin-6 in response to exposure to titanium-aluminum-vanadium particles. At 6 and 48 hours, the highest concentration of titanium alloy particles (0.189% [vol/vol]) resulted in 7-fold and <em>16</em>-fold increases in interleukin-6 release, respectively, when compared with negative controls. Neither interleukin-1 beta nor tumor necrosis <em>factor</em>-alpha was detected in the culture medium at any particle concentration tested for both dermal and foreskin <em>fibroblasts</em>. The pattern of prostaglandin E2 release by <em>fibroblasts</em> mirrored the pattern of interleukin-6 release. <em>Fibroblasts</em> exposed to the highest concentration of titanium alloy particles showed an increase in collagenase activity, starting at 12 hours. When medium samples were treated with amino phenylmercuric acetate to activate latent enzymes, a statistically significant increase in collagenase activity was observed as early as 6 hours (p < 0.001). Substrate gel analysis of medium from <em>fibroblasts</em> stimulated by high particle concentrations also showed an increase in gelatinolytic activity when compared with unstimulated controls. Analysis of medium samples for <em>growth</em> <em>factors</em> showed an increase in basic <em>fibroblast</em> <em>growth</em> <em>factor</em> at low particle concentrations, beginning at 12 hours. Levels of platelet-derived <em>growth</em> <em>factor</em>-AB and transforming <em>growth</em> <em>factor</em>-beta were not detectable in the controls or at any particle concentration tested. The results of this study showed that <em>fibroblasts</em> exposed to titanium alloy wear particles become activated and release proinflammatory mediators that influence bone metabolism. These data support the hypothesis that direct activation of <em>fibroblasts</em> by particulate wear may play a role in particle-mediated osteolysis. <em>Fibroblast</em> activation coupled with the biologic response of macrophages to wear debris in the loosening membrane may have a synergistic effect on pathologic bone resorption.

Hepatocyte <em>growth</em> <em>factor</em> (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-<em>16</em> to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in <em>fibroblast</em> cells.

BACKGROUND

Angiogenesis plays a significant role in the growth and progress of cancer, thus we evaluated the levels of urinary basic fibroblast growth factor (bFGF) in bladder cancer (Ca) patients and investigated any possible correlation between this angiogenic factor with tumor stage and grade.

METHODS

Urine samples from 41 patients with bladder Ca, 11 patients with history of bladder Ca but negative follow-up cystoscopy, 18 patients with benign prostate hyperplasia (BPH) and 15 normal healthy volunteers were assayed using an enzyme-linked immunosorbent assay for bFGF. Resulting values were normalized against urine creatinine and expressed as pg/g.

RESULTS

The median urinary bFGF level of patients with active disease, history of bladder carcinoma and negative follow-up cystoscopy, BPH, and healthy volunteers were 2,717, 1,009, 1,414 and 1,100 pg/g, respectively. There was a statistically significant difference between median bFGF levels of patients with active bladder Ca and those of the other groups (p = 0.000). Eleven patients with invasive bladder Ca had a median bFGF value of 6,880 pg/g that was significantly increased (p = 0.002) compared to the median of 2,312 pg/g of those with superficial tumors (Ta 14, T1 16). Grades 1, 2 and 3 carcinoma were found in 5, 19 and 17 patients which had a median bFGF of 2,717, 1,762 and 3,617 pg/g, respectively, but the difference was not statistically significant (p = 0.13).

CONCLUSIONS

Our results confirm the implication of bFGF in the biology of bladder cancer, and demonstrate that urinary bFGF concentration seems to be significantly related to the stage but not to the grade of the disease supporting the proposed mechanisms of release of bFGF. Further studies are required in order to validate the potential clinical applications of bFGF for specific groups of bladder cancer patients.