Manipulation of outer root sheath cell survival perturbs the hair-growth cycle.
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Transgenic mice that overexpress the anti-apoptotic gene bcl-xL under the control of the keratin 14 promoter have significantly shorter hair than non-transgenic littermates. The deficit in hair length correlated with a decrease in the duration of anagen, the growth phase of the hair cycle. A prolongation in telogen, the resting phase of the hair cycle, was also observed in adult animals. In the developing hair bulb, bcl-xL transgene expression was observed exclusively in the outer root sheath (ORS) cells. Bcl-xL expression enhanced the survival of ORS cells treated with apoptotic stimuli. The results suggest that preventing the apoptotic death of ORS cells during anagen leads to a more rapid termination of progenitor cell commitment/proliferation, while the increased survival of ORS cells during telogen delays the initiation of a new hair cycle. ORS cells produce fibroblast growth factor-5 (FGF-5), which acts in a paracrine fashion to terminate precursor cell division during anagen. The short hair phenotype of bcl-xL transgenic mice was substantially reversed in FGF-5-deficient mice. Thus, the production of growth inhibitory factors by ORS cells may provide a mechanism through which the hair-growth cycle is regulated by cell survival. (+info)
Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro.
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We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells. (+info)
The mRNA phenotype of a human RPE cell line at replicative senescence.
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PURPOSE: To explore the changes in expression of a set of genes in a single retinal pigment epithelial (RPE) cell line and two fibroblast cell lines as controls under culture conditions previously used for the analysis of senescent gene expression. METHODS: A single human RPE cell line, which had previously been characterized using known markers of senescence, and two fibroblast cell lines were grown to replicative exhaustion. The mRNA phenotype of genes known to be altered by senescence were studied by quantitative Northern analysis. RESULTS: The mRNA phenotype of cells changes at replicative senescence yielding a synthetic phenotype which is similar to cells found in repairing wounds. Of the genes studied, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 were regulated in RPE cells similar to fibroblasts at senescence. The largest changes noted for any single gene were the upregulation of insulin growth factor binding protein 2, and the downregulation of collagen I alpha 2, basic fibroblast growth factor, and fibroblast growth factor-5. CONCLUSIONS: This study demonstrates an altered mRNA phenotype of a human RPE cell line grown to replicative exhaustion. This analysis of a single cell line emphasizes the variability of results based on a single cell line or tissue specimen and indicates the need for additional study. (+info)
Dual-mode regulation of hair growth cycle by two Fgf-5 gene products.
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As the result of alternative mRNA splicing, Fgf-5, the gene encoding fibroblast growth factor-5, translates to both long and short forms of the protein, respectively, designated fibroblast growth factor-5 and fibroblast growth factor-5S. We previously showed that localization of fibroblast growth factor-5 and the level of fibroblast growth factor-5S in murine skin are hair-cycle dependent. In this study, we examined the effect of fibroblast growth factor-5 and fibroblast growth factor-5S on the hair growth cycle in mice. Once the anagen phase of the hair growth cycle was induced in the dorsal skin by depilation during telogen, and effects of subcutaneous injection of fibroblast growth factor-5 and fibroblast growth factor-5S into the affected region were analyzed. We found that fibroblast growth factor-5 inhibited hair growth during anagen and promoted the transition from anagen to catagen. Interestingly, whereas fibroblast growth factor-5S alone exerted no effect on hair growth, it significantly inhibited the catagen-promoting activity of fibroblast growth factor-5 when the two proteins were injected simultaneously. Because neither fibroblast growth factor-5 nor fibroblast growth factor-5S affected skin thickness, it is postulated that changes in skin thickness during hair cycle are separately regulated by factors other than those regulating hair and follicle growth. The present results, together with our earlier findings that fibroblast growth factor-5-producing cells gather around dermal papillae during catagen, whereas fibroblast growth factor-5S is abundantly expressed in the hair follicles only during the latter half of anagen, suggests that the mouse hair growth cycle is regulated by the two Fgf-5 gene products acting in concert: fibroblast growth factor-5 induces catagen, whereas fibroblast growth factor-5S antagonizes this activity during anagen. (+info)
Differential display identification of 40 genes with altered expression in activated human smooth muscle cells. Local expression in atherosclerotic lesions of smags, smooth muscle activation-specific genes.
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Detailed knowledge on the molecular and cellular mechanisms that control (re)-differentiation of vascular smooth muscle cells (SMCs) is critical to understanding the pathological processes underlying atherogenesis. We identified by differential display/reverse transcriptase-polymerase chain reaction 40 genes with altered expression in cultured SMCs upon stimulation with the conditioned medium of activated macrophages. This set of genes comprises 10 known genes and 30 novel genes, which we call "smags" (for smooth muscle activation-specific genes). To determine the in vivo significance of these (novel) genes in atherogenesis, we performed in situ hybridization experiments on vascular tissue. Specifically, FLICE (Fas-associated death domain-like interleukin-1beta-converting enzyme)-like inhibitory protein (FLIP) is expressed in neointimal SMCs as well as in lesion macrophages and endothelial cells, whereas the expression of the novel genes smag-63, smag-64, and smag-84 is restricted to neointimal SMCs. Characterization of full-length smag-64 cDNA revealed that it encodes a novel protein of 66 amino acids. smag-82 cDNA comprises the complete, unknown, 3'-untranslated region of fibroblast growth factor-5. Collectively, our results illustrate the complex changes of SMC gene expression that occur in response to stimulation with cytokines and growth factors secreted by activated macrophages. Moreover, we identified interesting candidate genes that may play a role in the differentiation of SMCs during atherogenesis. (+info)
FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb.
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FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS-FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin-expressing, connective-tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked-cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle. (+info)
Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of FGF-5 and FGF-18.
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The goal of these experiments was to evaluate the potential of the fibroblast growth factor family members FGF-5 and FGF-18 to rescue photoreceptors from cell death in retinal degenerative disease. Two strains of transgenic rats, expressing either a P23H or an S334ter rhodopsin mutation, were used as model systems. The neurotrophic growth factors were delivered by subretinal injection of adeno-associated virus vectors, driving expression of the genes with a constitutive CMV promoter. Morphological and functional analyses were performed to determine whether FGF-5 or FGF-18 overexpression could ameliorate cell death in the retina. Immunocytochemistry was used to determine the cellular sites of expression of the factors and to test for up-regulation of FGF receptors due to injection. Significant rescue from photoreceptor cell death was found after injections of vectors expressing either FGF-5 or FGF-18 in the animal models. Increased survival of photoreceptors did not produce a significant increase in electroretinographic responses, however, reflecting either trauma due to the surgery or a suppression of signaling due to expression of proteins. Three weeks after injections, both growth factors were localized to the inner and outer segments of photoreceptors, and the receptors FGFR1 and FGFR2 were also found to be up-regulated in these regions. No visible pathological changes were seen in the FGF-5- or FGF-18-treated eyes. These results indicate that the delivery of either FGF-5 or FGF-18 with adeno-associated virus protects photoreceptors from apoptosis in transgenic rat models of retinitis pigmentosa and that the rescue is probably mediated by conventional receptor tyrosine kinase pathways in photoreceptors. (+info)
Identification of fibroblast growth factor-5 as an overexpressed antigen in multiple human adenocarcinomas.
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Methodology for identifying tumor-associated antigens recognized by T cells has been successfully used to clone antigens from melanoma cells. Similar efforts for nonmelanoma tumors have had limited success with few antigens identified. To identify potentially relevant tumor-associated antigens expressed in renal cell carcinoma cell lines, a tumor-specific CTL clone was established from tumor-infiltrating lymphocytes from a regressing pulmonary lesion. This CTL recognized nonmutated fibroblast growth factor-5 (FGF-5). Quantitative real-time reverse transcription PCR revealed that FGF-5 was overexpressed in the majority of renal cell carcinomas, as well as in some prostate carcinoma and breast carcinoma lines. FGF-5 expression by quantitative real-time reverse transcription PCR in normal tissues was below the recognition threshold for this CTL. As a normal protein with significant overexpression by multiple adenocarcinomas and little normal tissue expression, FGF-5 represents an immunotherapy target with potential utility against a broad array of nonmelanoma cancers. (+info)