Prolyl 4-hydroxylase alpha-related proteins in Drosophila melanogaster: tissue-specific embryonic expression of the 99F8-9 cluster.
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The extracellular matrix (ECM) is proposed to play critical roles in organ morphogenesis through the stabilization and/or sequestration of signaling factors and adhesion molecules, and by maintaining organ integrity. As a first step toward understanding molecules involved in ECM modification and maturation, we have examined the embryonic expression profiles of ten prolyl 4-hydroxylase alpha subunit (PH4alpha)-related genes. Prolyl 4-hydroxylases (PH4) catalyze the formation of 4-hydroxyproline in collagens, the major components of the ECM, and are implicated in the hydroxylation of proline in several other secreted proteins. To date, two alpha subunit proteins have been described in both humans (PHalphaI and PHalphaII) and worms (PHY-1/DPY-18 and PHY-2), whereas only a single Drosophila alpha subunit has been identified. The ten PH4alpha-related genes described in this study are clustered in a 183-kb region near the tip of chromosome arm 3R and include the previously described Drosophila alpha subunit gene. Six of the ten PH4alpha genes in the cluster have tissue-specific embryonic expression. PH4alphaSG1 and PH4alphaSG2 are expressed in the salivary gland, PH4alphaMP is expressed in mouth-part precursors, PH4alphaPV is expressed in the proventriculus, and CG9698-E is expressed in the epidermis. PH4alphaEFB is expressed more broadly, with expression in the anterior and posterior midgut primordia, the fat body, the hemocytes and the epidermis. The expression profiles of these PH4alpha-related genes suggest that tissue-specific ECM modifications may be critical to organ formation and/or function. (+info)
Novel estrogen and tamoxifen induced genes identified by SAGE (Serial Analysis of Gene Expression).
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The breast cancer promoting effects of estrogen and the chemopreventive effects of tamoxifen are thought to be mediated by the estrogen receptor, a ligand-dependent transcription factor. Therefore, comprehensive analysis of gene expression profiles following estrogen or tamoxifen treatment may help us better understand the role estrogen plays in tumorigenesis. We utilized SAGE (Serial Analysis of Gene Expression) technology to identify genes regulated by estrogen and tamoxifen in the ZR75-1 estrogen dependent breast cancer cell line. In this manner we have identified several genes that were regulated by estrogen or tamoxifen. Here we report the identification and initial characterization of EIT-6 (Estrogen Induced Tag-6), a novel nuclear protein and a new member of the evolutionarily conserved SM-20 family of growth regulatory immediate-early genes. EIT-6 appears to be a direct transcriptional target of the estrogen receptor and constitutive expression of EIT-6 promotes colony growth in human breast cancer cells. These data indicate that EIT-6 may play a role in estrogen induced cell growth. (+info)
Egg shell collagen formation in Caenorhabditis elegans involves a novel prolyl 4-hydroxylase expressed in spermatheca and embryos and possessing many unique properties.
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The collagen prolyl 4-hydroxylases (EC ) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are alpha(2)beta(2) tetramers, in which the beta subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic alpha subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. We report here on the cloning and characterization of a third C. elegans alpha subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans alpha subunits and shows 23-30% amino acid sequence identity to PHY-1 and PHY-2 within the catalytic C-terminal region. Recombinant PHY-3 coexpressed in insect cells with a C. elegans PDI isoform that does not associate with PHY-1 was found to be an active prolyl 4-hydroxylase. The phy-3 gene consists of five exons, and its expression pattern differs distinctly from the hypodermally expressed phy-1 and phy-2 in that it is expressed in embryos, late larval stages, and adult nematodes, expression in the latter being restricted to the spermatheca. Nematodes homozygous for a phy-3 deletion are phenotypically of the wild type and fertile, but the 4-hydroxyproline content of phy-3(-/-) early embryos was reduced by about 90%. PHY-3 is thus likely to be involved in the synthesis of collagens in early embryos, probably of those in the egg shell. (+info)
Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant.
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Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132-136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C. (+info)
Cloning and characterization of a low molecular weight prolyl 4-hydroxylase from Arabidopsis thaliana. Effective hydroxylation of proline-rich, collagen-like, and hypoxia-inducible transcription factor alpha-like peptides.
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4-Hydroxyproline is found in collagens and collagen-like proteins in animals and in many glycoproteins in plants. Animal prolyl 4-hydroxylases (P4Hs) have been cloned and characterized from many sources, but no plant P4H has been cloned so far. We report here that the genome of Arabidopsis thaliana encodes six P4H-like polypeptides, one of which, a 283-residue soluble monomer, was cloned and characterized here as a recombinant protein. Catalytically critical residues identified in animal P4Hs are conserved in this P4H, and their mutagenesis led to complete or almost complete inactivation. The recombinant P4H effectively hydroxylated poly(l-proline) and many synthetic peptides corresponding to proline-rich repeats present in plant glycoproteins and other proteins. Surprisingly, collagen-like peptides were also good substrates, the V(max) with (Pro-Pro-Gly)(10) being similar to that with poly(l-proline). The enzyme acted in this peptide preferentially on prolines in Y positions in the X-Y-Gly triplets. Correspondingly, (Gly-Pro-4Hyp)(5) and (Pro-Ala-Gly)(5) were poor substrates, with V(max) values less than 5 and 20% of that obtained with (Pro-Pro-Gly)(10), respectively, the K(m) for the latter also being high. Peptides representing the N- and C-terminal hydroxylation sites present in hypoxia-inducible transcription factor alpha also served as substrates. As these peptides contain only one proline residue, a poly(l-proline) type II conformation was clearly not required for hydroxylation. (+info)
Cloning and characterization of the 5'-flanking region of the rat P4Halpha gene encoding the prolyl 4-hydroxylase alpha(I) subunit.
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Prolyl 4-hydroxylase (P4H), an alpha(2)beta(2) tetramer, plays a crucial role in collagen synthesis. It catalyzes the hydroxylation of proline residues in X-Pro-Gly sequences to form 4-hydroxyproline. We isolated a genomic clone of the rat P4Halpha(I) gene. Approx. 0.6 kb of the fragment, which contained the 5'-flanking region, exon 1, and intron 1, was sequenced. Computer analysis revealed several motifs that may act as binding sites for basal transcription factors. To elucidate the regulation of the rat P4Halpha(I) gene expression, we assessed the 0.6 kb 5'-flanking region of the P4Halpha(I) gene for basal promoter activity. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. The basal expression level of these constructs was determined in fetal rat lung fibroblasts and Hepa-1 hepatoma cells. By measuring the luciferase activity, we found a positive-regulatory region at positions -246 to -165 bp. (+info)
The exoskeleton collagens in Caenorhabditis elegans are modified by prolyl 4-hydroxylases with unique combinations of subunits.
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The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly. (+info)
Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies.
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BACKGROUND: In recent studies performed on cultured cells and experimental nephropathies, it has been hypothesized that tubular epithelial cells (TEC), via epithelial-mesenchymal transformation (EMT), can become collagen-producing cells. According to this theory, they should proceed through several activating steps, such as proliferation and phenotype changes, to eventually synthesize extracellular matrix (ECM). METHODS: To evaluate whether EMT operates in human TECs, 133 renal biopsies of different renal diseases were studied, analyzing by immunohistochemistry and in situ hybridization the possible expression of markers of proliferation (PCNA, Mib-1), cellular phenotype (vimentin, alpha-SMA, cytokeratin, ZO-1) and ECM production (prolyl 4-hydroxylase, HSP47, interstitial collagens). RESULTS: Independently of histological diagnosis, variable degrees of TEC positivity for PCNA (2.7 +/- 2.4 cells/field) and Mib-1 (1.9 +/- 2.3) were present. TECs expressing vimentin (1.4 +/- 4.7) and alpha-smooth muscle actin (alpha-SMA; 0.04 +/- 0.4) also were detected. It was possible to observe loss of epithelial antigens from 8 to 10% of the tubular cross sections. Moreover, TECs were stained by prolyl 4-hydroxylase (3.6 +/- 4.3), heat shock protein-47 (HSP47; 2.9 +/- 5.4), collagen type I (0.2 +/- 2.7) and type III (0.3 +/- 2.0). Collagen types I and III mRNAs were found in 0.8 to 1.4 cells/field. The number of TEC with EMT features were associated with serum creatinine and the degree of interstitial damage (P< or = 0.03), and even considering the 45 cases with mild interstitial lesions, the tubular expression of all markers remained strictly associated with renal function (P< or = 0.01). CONCLUSIONS: Our results suggest that, via transition to a mesenchymal phenotype, TEC can produce ECM proteins in human disease and directly intervene in the fibrotic processes. Moreover, the association of EMT features with serum creatinine supports the value of these markers in the assessment of disease severity. (+info)