Regulation of α1(I) Collagen Messenger RNA Decay by Interactions with αCP at the 3′-Untranslated Region

Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein αCP binds to the α1(I) collagen 3′-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of αCP in the decay of transcripts containing the collagen 3′-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, αCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of αCP binding activity increased the rate of decay of wild type transcripts containing the αCP 3′-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no αCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for αCP. The addition of recombinant αCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain αCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting αCP showed a reduction of the binding activity of αCP and a concomitant reduction in intracellular levels of α1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of αCP in the stabilization of α1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs. Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein αCP binds to the α1(I) collagen 3′-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of αCP in the decay of transcripts containing the collagen 3′-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, αCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of αCP binding activity increased the rate of decay of wild type transcripts containing the αCP 3′-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no αCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for αCP. The addition of recombinant αCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain αCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting αCP showed a reduction of the binding activity of αCP and a concomitant reduction in intracellular levels of α1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of αCP in the stabilization of α1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs. Hepatic fibrosis is a result of excess deposition of extracellular matrix proteins, including type I collagen, by activated hepatic stellate cells (HSCs) 1The abbreviations used are: HSC, hepatic stellate cell; UTR, untranslated region; mRNA, messenger RNA; WT, wild type; MUT, mutant; siRNA, small interfering RNA; poly(A), polyadenylated; PABP, poly(A)-binding protein; ARE, AU-rich element; EMSA, electrophoretic mobility shift assay; IVDA, in vitro decay assay. (1Friedman S.L. Rockey D.C. McGuire R.F. Maher J.J. Boyles J.K. Yamasaki G. Hepatology. 1992; 15: 234-243Crossref PubMed Scopus (244) Google Scholar, 2Friedman S.L. Alcohol. Clin. Exp. Res. 1999; 23: 904-910PubMed Google Scholar, 3Schuppan D. Semin. Liver Dis. 1990; 10: 1-10Crossref PubMed Scopus (327) Google Scholar). As a general response to most chronic stimuli, HSCs are transformed from a quiescent to that of an activated phenotype (4Aycock R.S. Seyer J.M. Connect. 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The increase in α1(I) collagen expression is primarily because of an increase in the half-life of the α1(I) collagen mRNA molecule, from 1.5 h in quiescent HSCs to greater than 24 h in activated HSCs (9Lindquist J.N. Stefanovic B. Brenner D.A. J. Gastroenterol. 2000; 35: 80-83PubMed Google Scholar, 10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar). The increase in α1(I) collagen mRNA stability coincides with an increase in the binding activity of αCP (hnRNP E2, PCBP2) to a C-rich region in the α1(I) collagen 3′-untranslated region (UTR) (10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar). 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Celis J.E. Eur. J. Biochem. 1995; 230: 447-453Crossref PubMed Scopus (195) Google Scholar). Yeast two-hybrid studies have demonstrated that αCP may form homodimers and have identified other nucleic acid-binding proteins with which it may interact, such as poly(A)-binding protein (PABP) (13Funke B. Zuleger B. Benavente R. Schuster T. Goller M. Stevenin J. Horak I. Nucleic Acids Res. 1996; 24: 3821-3828Crossref PubMed Scopus (38) Google Scholar, 26Wang Z. Kiledjian M. Mol. Cell. Biol. 2000; 20: 6334-6341Crossref PubMed Scopus (86) Google Scholar). Regulated RNA decay can be analyzed in vitro by following a radiolabeled RNA containing the sequence of interest over time in cytoplasmic extracts (26Wang Z. Kiledjian M. Mol. Cell. Biol. 2000; 20: 6334-6341Crossref PubMed Scopus (86) Google Scholar, 27Ford L.P. Wilusz J. Methods. 1999; 17: 21-27Crossref PubMed Scopus (53) Google Scholar, 28Ford L.P. Watson J. Keene J.D. Wilusz J. 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A. 2002; PubMed Scopus Google Scholar, Genes Dev. 2002; PubMed Scopus Google Scholar). that the in vitro decay in vivo used to αCP levels in activated HSCs and observed this on collagen α1(I) This study utilized from quiescent and activated HSCs to demonstrate that the stability of the α1(I) collagen mRNA is directly mediated by binding of αCP to binding in the expression of αCP in HSCs in α1(I) collagen mRNA a direct role for αCP in the stabilization of α1(I) collagen in activated HSCs. Cell cells in with HSCs as H. Brenner D.A. Hepatology. 1996; 24: PubMed Google Scholar). of NIH or time in of and the The cells and incubated in a 1.5 on and cells with a was by and the as cytoplasmic from the cytoplasmic by in a for in a was to a of and the and wild type collagen α1(I) 3′-UTR was and to in vitro transcription with the The mutant 3′-UTR (10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google that αCP binding was the to the The addition of to the of the 3′-UTR was performed as L.P. Wilusz J. Methods. 1999; 17: 21-27Crossref PubMed Scopus (53) Google Scholar). The containing the sequence was the the and a and the transformed and and radiolabeled from 3′-UTR and was to an in vitro transcription containing of of with of and of RNA in a containing and radiolabeled from the and in radiolabeled of and of kinase with T. J. a Scholar). In vitro decay assays performed as L.P. Wilusz J. Methods. 1999; 17: 21-27Crossref PubMed Scopus (53) Google Scholar). the decay was performed in a containing and radiolabeled and of a radiolabeled as a for and the and to a and of was The was on of of and the of the was and the RNA with of RNA was in of and on a containing recombinant was as Biochem. 1993; PubMed Scopus Google Scholar, J.N. Stefanovic B. Brenner D.A. Nucleic Acids Res. 2000; PubMed Google Scholar). was from the in by with for h to protein was cell with the addition of of a of The three with and recombinant protein reduced in of of the different time was the The time was as of the and for by to the of the radiolabeled in the experiments three RNA RNA from HSCs was as H. Brenner D.A. Hepatology. 1996; 24: PubMed Google Scholar). the to the was from HSCs with in with of in a of following the analyzed h RNA or extracts as of used to to in a mobility shift assays performed as J.N. Stefanovic B. Brenner D.A. Nucleic Acids Res. 2000; PubMed Google Scholar). of cytoplasmic incubated in a binding with of for on in and by of activated HSC extracts incubated with of quiescent extracts or as a as with by and incubated with and incubated with αCP a from M. of RNA in in NIH study the involved in α1(I) collagen mRNA utilized radiolabeled and transcripts as RNA and protein extracts in J.E. H. Trends Biochem. Sci. 1995; 20: Full Text PDF PubMed Scopus Google Scholar, J. 1995; PubMed Google Scholar). studies to a regulated decay of the collagen 3′-UTR utilized extracts from NIH cells that type I collagen and the binding activity in activated HSCs from NIH cells and activated HSCs and HSC, but not quiescent HSCs have binding activity for the collagen This binding activity been identified as αCP assays with J.N. Stefanovic B. Brenner D.A. Nucleic Acids Res. 2000; PubMed Google Scholar). RNA decay a in NIH an in vitro decay containing of was The addition of excess to the increase the decay rate decay by a (26Wang Z. Kiledjian M. Mol. Cell. Biol. 2000; 20: 6334-6341Crossref PubMed Scopus (86) Google Scholar, Z. N. P. Kiledjian M. Mol. Cell. Biol. 1999; 19: PubMed Scopus (219) Google Scholar). The addition of of increased the rate of decay of the whereas addition of other on the rate of decay a that the decay a the of the RNA to be by αCP on the rate of decay in the NIH The decay of the which αCP and the which αCP to NIH demonstrated an increase in stability of the compared with the or in a is of the transcript in NIH of the α1(I) collagen 3′-UTR is with that for the α-globin mRNA and the that αCP can directly the α1(I) collagen Competition of αCP that αCP binding to the α1(I) collagen 3′-UTR RNA the decay of the mRNA, studies performed an binding sequence for αCP. the αCP binding is by the addition of observed an increase in the decay rate of the 3′-UTR transcript to a rate very to the 3′-UTR transcript and The addition of a mutant of the of binding αCP no on the decay rate of the 3′-UTR and the the 3′-UTR lacking the αCP binding showed a increase in the rate of decay with the but not the This may αCP from the transcript because of a direct of with αCP (26Wang Z. Kiledjian M. Mol. Cell. Biol. 2000; 20: 6334-6341Crossref PubMed Scopus (86) Google Scholar, Z. N. P. Kiledjian M. Mol. Cell. Biol. 1999; 19: PubMed Scopus (219) Google Scholar). that αCP binding to the α1(I) collagen 3′-UTR transcript stability in a of and in Quiescent HSCs that the in vitro decay in vivo RNA the stability of and transcripts was in from quiescent HSC extracts αCP binding activity expression of αCP protein (10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar). αCP binding activity is to the collagen mRNA the the decay of the and 3′-UTR transcripts be in As the 3′-UTR and the 3′-UTR decay This demonstrates the that αCP to the 3′-UTR to the transcript, as observed in NIH extracts in a difference in decay the and 3′-UTR transcripts in activated performed extracts obtained from HSCs. A increase in the stability of the transcript was observed compared with the transcript and αCP of αCP the stability of the 3′-UTR recombinant αCP was to the quiescent HSC extracts that do not contain an αCP binding activity αCP the 3′-UTR transcript but to the stability of the 3′-UTR transcript The increase in the but not transcript a direct role for the binding of αCP in the α1(I) collagen of αCP α1(I) mRNA the role of αCP in targeting the αCP mRNA cells that α1(I) collagen and αCP binding of the αCP as by EMSA, was by addition of αCP compared with a the the binding activity of to the of αCP binding activity on the levels of the α1(I) collagen levels of α1(I) collagen mRNA reduced in cells with αCP compared with as by was no in mRNA the role of αCP in the α1(I) collagen the of αCP expression in αCP HSCs and αCP binding by A of in αCP binding activity was observed in HSCs with targeting αCP compared with the levels of α1(I) collagen mRNA in HSCs with αCP and observed a in the α1(I) collagen mRNA levels experiments demonstrate that αCP with the α1(I) collagen mRNA 3′-UTR stability of the mRNA in activated HSCs. of αCP in the of αCP binding HSC the of αCP protein in quiescent and activated HSCs. In quiescent HSC αCP was primarily in the with very αCP protein levels observed in the cytoplasmic in the activated HSC extracts the of αCP protein expression increased and a of αCP to the cytoplasmic activated HSCs contain αCP protein and the αCP protein demonstrates a markedly different the binding activity of was in extracts from quiescent and activated HSCs not the of binding activity observed in quiescent HSCs is because of levels of protein expression in the or is of αCP binding in the quiescent binding activity in of activated HSC extracts with of no on the observed binding the addition of of quiescent HSC extracts reduced the binding activity of the activated HSC extracts by in the cytoplasmic In the the binding activity was not activation of HSCs is by an increase in the levels of α1(I) collagen mRNA (10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar, D.A. L. R. J.M. Hepatology. 1993; 17: Google Scholar). This increase results primarily from an increase in the half-life of the α1(I) collagen mRNA from 1.5 to The increased half-life of the α1(I) collagen mRNA with increased binding activity of αCP to the α1(I) collagen 3′-UTR following HSC activation (10Stefanovic B. Hellerbrand C. Holcik M. Briendl M. Aliebhaber S. Brenner D.A. Mol. Cell. Biol. 1997; 17: 5201-5209Crossref PubMed Google Scholar). This study demonstrates that αCP a direct role in of the α1(I) collagen mRNA in activated, but not quiescent, HSCs. proteins can be for role in mRNA degradation or RNAs that in αCP binding sites different decay with NIH cell a role for αCP in mRNA Competition of αCP binding an results in a decay of transcripts containing a αCP binding site, a direct role of αCP in mRNA the role of αCP in the of α1(I) collagen mRNA expression in quiescent and activated HSCs. Quiescent HSCs of type I collagen, the α1(I) collagen mRNA a and the cells contain no αCP binding activity S.L. Alcohol. Clin. Exp. Res. 1999; 23: 904-910PubMed Google Scholar, 5Safadi R. Friedman S.L. MedGenMed. 2002; 4: 27PubMed Google Scholar, H. Brenner D.A. Hepatology. 1996; 24: PubMed Google Scholar, J.N. Stefanovic B. Brenner D.A. Nucleic Acids Res. 2000; PubMed Google Scholar). The decay with quiescent extracts for and 3′-UTR RNA are as in that αCP binding and 3′-UTR RNAs incubated with from activated which produce collagen, have a half-life of the α1(I) collagen mRNA, and contain αCP binding a in the decay rate was This that αCP binding activity is for the α1(I) collagen mRNA by the 3′-UTR in activated HSCs. different extracts be directly the rate of decay of different transcripts in the of the and rate of decay because are not a binding activity of αCP in activated, but not quiescent to the increase of the collagen mRNA As a to the role of αCP binding in the α1(I) collagen mRNA, was αCP binding activity was by in activated HSCs with siRNA, whereas a no on αCP binding This reduction of αCP binding activity in mRNA levels of α1(I) collagen, whereas cells with demonstrate that the increased αCP binding activity in activated HSCs is for α1(I) collagen mRNA stability resulting in mRNA αCP protein is from primarily a to to the and the HSC This αCP to to the collagen α1(I) 3′-UTR and the mRNA from demonstrate that the quiescent HSC contains an activity that αCP binding activity in activated cytoplasmic the other to in αCP to the in binding such as phosphorylation, or not on (10Stefanovic B. 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HSCs increase the stability of the α1(I) collagen mRNA as the cells αCP binding to a region in the 3′-UTR of α1(I) collagen is for increased stability of the α1(I) collagen liver quiescent HSCs a and vitamin A and increase of extracellular matrix proteins, including α1(I) the of type I collagen in this have demonstrated that transcripts are by the of an αCP binding in activated, but not quiescent HSC addition of recombinant αCP stability to transcripts containing αCP binding sites in quiescent HSC of αCP results in αCP binding activity and a loss of collagen α1(I) mRNA the activation of HSCs a of αCP from primarily in quiescent HSCs to cytoplasmic and in activated and is a cytoplasmic activity in quiescent HSCs that the binding activity of activated αCP. this demonstrates for the time that αCP binding activity is for collagen α1(I) mRNA stabilization HSC activation and that of this activity is sufficient to the collagen α1(I) mRNA αCP is a for of liver the of and for HSC