Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3' untranslated region in pro alpha 1 (I) collagen cDNA.

Herget, Thomas; Burba, M; Schmoll, Marion; Zimmermann, Katrin; Starzinski‐Powitz, Anna

doi:10.1128/mcb.9.7.2828

Cited by 19 publications

(9 citation statements)

References 38 publications

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“…From the Northern/dot blot double-hybridization experiment, the relative amounts of Cx40 and Cx37 transcript in hm& were estimated and used to standardize the results of both Northern blot hybridizations. The amounts of poly(A) + RNA on Northern blots were estimated by hybridization to the 1.58-kb Ez, oRI-SalI fragment of mouse cytochrome c oxidase eDNA (clone pAG82; Herget et al, 1989).…”

Section: Southern and Northern Blot Analysismentioning

confidence: 99%

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung

Hennemann

Suchyna

Lichtenberg‐Fraté

et al. 1992

The Journal of Cell Biology

170

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Abstract.From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (or subgroup of connexins) than to mouse Cx32 (B subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lungsimilar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two-to fourfold higher than in the corresponding adult tissues.Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (r = 580 ms at Vj of +50 mY). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into couplingdeficient human I-IeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

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Section: Southern and Northern Blot Analysismentioning

confidence: 99%

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung

Hennemann

Suchyna

Lichtenberg‐Fraté

et al. 1992

The Journal of Cell Biology

170

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“…For example, stellate cell type collagen I mRNA is stabilized after wound healing and contributes to enhanced type I collagen mRNA and protein expression (Stefanovic et al, 1997(Stefanovic et al, , 1999. In these experiments, increased mRNA stability was a result of ␣CP binding to poly(C)-rich areas of the type I collagen 3Ј UTR mRNA (Focht and Adams, 1984;Herget et al, 1989;Stefanovic et al, 1997Stefanovic et al, , 1999. Thus, our work has extended the concept that hepatic wound healing is associated with changes in mRNA stability by identifying TGF-␤, a critical cytokine involved in diverse forms of wounding (Border and Noble, 1994;Sanderson et al, 1995), as a regulator of 3Ј UTR binding proteins that contribute to mRNA stability during this process.…”

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confidence: 95%

Cell and Molecular Regulation of Endothelin-1 Production during Hepatic Wound Healing

Shao

Shi

Gotwals

et al. 2003

MBoC

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During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelinconverting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56-and 62-kDa ECE-1 3Ј-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3Ј UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3Ј UTR mRNA (62 kDa). A functional role for the 3Ј UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-␤1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56-and 62-kDa ECE-1 3Ј UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56-and 62-kDa proteins, which in turn are regulated by transforming growth factor-␤. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised. INTRODUCTIONThe response to hepatic wounding is characterized by the activation of hepatic stellate cells (also lipocytes, fat-storing cells), critical effectors of the wound healing process (Ballardini et al., 1988;Friedman and Arthur, 1989;Kent et al., 1976;Maher and McGuire, 1990). During stellate cell activation, these cells exhibit features of myofibroblasts, producing increased quantities of extracellular matrix proteins as well as smooth muscle ␣-actin. A further characteristic of stellate cell activation is enhanced production of the vasoconstrictive peptide, endothelin-1 (ET-1), which contributes to perpetuation of the fibrogenic process (Gandhi et al., 1998;Rockey and Chung, 1996) as well as in control hepatic vascular tone (Bauer et al., 1994;Kawada et al., 1993;Rockey and Weisiger, 1996).Each of the three known endothelin isoforms (-1, -2, -3) arise by proteolytic processing of large precursors (ϳ200 amino acid residues). Intermediates, termed big ET-1, -2, and -3 (38 -41 aa) are excised from prepropeptides at sites containing paired basic amino acids. Big endothelins, which have little or no biological activity (Yanagisawa, 1994), are cleaved at Trp-21-Val/Ile-22 to produce mature 21-residue, biologically active peptides. The enzyme responsible for the specific cleavage at Trp-21 has been termed endothelinconverting enzyme (ECE); it is a neutral membrane-bound metalloprotease with M r ϭ 120 kDa, belonging to the endopeptidase-24.11 family found in brain (Ohnaka et al., 1993;...

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“…Contribution of the COL1A1 Minigene 3Ј-Flanking Sequence-Several groups have examined the role of the COL1A1 3Ј-flanking and untranslated (3Ј-UTR) regions in transcriptional regulation as well as mRNA stabilization (32,33). Recently, Sokolov et al (27) reported that 90% of the 3Ј-UTR, in the context of the human COL1A1 minigene, was not essential for tissue-specific expression.…”

Section: Discussionmentioning

confidence: 99%

COL1A1 Transgene Expression in Stably Transfected Osteoblastic Cells

Breault

Lichtler

1997

Journal of Biological Chemistry

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Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50 -100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.Type I collagen is the most abundant protein in vertebrates with levels reaching 5-65% of total protein synthesis in bone cells (1, 2). Two coordinately regulated genes, COL1A1 and COL1A2, give rise to the mRNA transcripts, each containing 50 -51 exons, which are processed, translated, and post-transcriptionally modified. The resulting procollagen chains are assembled into a heterotrimeric structure and modified further before entering into the collagen fibril, which in turn is modified by its interaction with other extracellular molecules. The primary site of regulation for this complex series of events is thought to be at the level of transcription initiation. Most studies aimed at understanding the transcriptional regulation of COL1A1 have focused on cell-specific regulatory domains within the proximal promoter, the first intron, and more distal 5Ј-upstream domains without attention to sequences accounting for the extremely high level of gene transcription (3-8).We have been studying the regulation of the rat COL1A1 gene utilizing a family of transgenes, designated ColCAT, which are derived from the expression vector pSV 2 CAT. These transgenes are expressed in a tissue-specific manner in bone, tendon, and skin of transgenic mice and have been used to elucidate a bone-specific regulatory ele...

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Regulated expression of nuclear protein(s) in myogenic cells that binds to a conserved 3' untranslated region in pro alpha 1 (I) collagen cDNA.

Cited by 19 publications

References 38 publications

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung

Cell and Molecular Regulation of Endothelin-1 Production during Hepatic Wound Healing

COL1A1 Transgene Expression in Stably Transfected Osteoblastic Cells

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