Nuclear matrix targeting of the protein kinase CK2 signal as a common downstream response to androgen or growth factor stimulation of prostate cancer cells.
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Protein kinase CK2, a messenger-independent serine/threonine kinase, has been implicated in cell growth. Androgenic stimulus in rat prostate modulates its association with nuclear matrix (NM) and chromatin. Because the growth of human prostate carcinoma cells is influenced by androgens and/or growth factors, we determined the nature of CK2 signaling in the NM in response to androgen and growth factor stimuli. Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were cultured in media to regulate their growth in the presence of 5alpha-dihydrotestosterone (5alpha-DHT) or growth factors (epidermal growth factor, keratinocyte growth factor, and transforming growth factor alpha). The activity of CK2 was measured in the cytosolic and NM fractions isolated from these cells after treatment with growth stimuli. The changes in CK2 in various fractions were also confirmed by immunoblotting with a specific antibody. LNCaP cells responded to both 5alpha-DHT and growth factors for growth. The presence of these agents in the culture medium evoked a translocation of CK2 to the NM from the cytosol. The PC-3 cells did not respond to 5alpha-DHT for growth but did respond to growth factors. Under these conditions, there was also a translocation of CK2 to the NM concomitant with a decrease in the cytosolic fraction. These results suggest that CK2 translocation to the NM occurs in response to various growth stimuli in cells in culture. Thus, CK2 is a common downstream signal transducer in response to diverse growth stimuli that may relate to the pathobiology of prostate cancer cells. (+info)
Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture.
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We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development. (+info)
Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells.
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Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF. (+info)
Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.
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A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic. (+info)
Extracellular matrix-dependent activation of syndecan-1 expression in keratinocyte growth factor-treated keratinocytes.
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Syndecan-1 is a major heparan sulfate proteoglycan of the epidermis. Its expression is strongly induced in migrating and proliferating keratinocytes during wound healing and, on the other hand, diminished or lost in invasive squamous cell carcinoma. We have recently found in the syndecan-1 gene an enhancer (fibroblast growth factor-inducible response element (FiRE)) that activates gene expression in wound edge keratinocytes (Jaakkola, P., Kontusaari, S., Kauppi, T., Maatta, A., and Jalkanen, M. (1998) FASEB J. 12, 959-969). Now, we demonstrate that the activation of this enhancer by keratinocyte growth factor (KGF) is modulated by the components of the extracellular matrix (ECM). MCA-3D mouse immortal keratinocytes growing on fibrillar collagen failed to activate FiRE and subsequently to induce syndecan-1 in response to KGF. The same cells growing on fibronectin or laminin, however, increased FiRE-dependent reporter gene expression upon KGF treatment. The inhibition of the KGF induction by collagen appears to be specific for signaling to FiRE, as the increase in cell proliferation by KGF was not affected. The effect was selective to KGF, as EGF-induction was independent on ECM composition. Changes in the transcription factor binding were not involved in the differential activation of FiRE, as the levels and composition of the AP-1 complexes were unchanged. However, application of anisomycin, an activator of Jun amino-terminal kinase, resulted in a lower response in cells growing on collagen compared with fibronectin. These results indicate that the composition of ECM and availability of growth factors can play a role in the epidermal regulation of syndecan-1 expression and that FiRE is a novel target for gene regulation by the extracellular matrix. (+info)
Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors.
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Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner. (+info)
Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice.
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Keratinocyte growth factor (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -2 and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs. (+info)
Phenotypic control of gap junctional communication by cultured alveolar epithelial cells.
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We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins. (+info)