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. 2005 Oct;16(10):4931-40.
doi: 10.1091/mbc.e05-03-0216. Epub 2005 Aug 10.

YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts

Aiwen Zhang  1 , Xiaoying Liu, John G Cogan, Matthew D Fuerst, John A Polikandriotis, Robert J Kelm Jr, Arthur R Strauch
Affiliations

YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts

Aiwen Zhang et al. Mol Biol Cell. 2005 Oct.

Abstract

Profibrotic regulatory mechanisms for tissue repair after traumatic injury have developed under strong evolutionary pressure to rapidly stanch blood loss and close open wounds. We have examined the roles played by two profibrotic mediators, transforming growth factor beta1 (TGFbeta1) and thrombin, in directing expression of the vascular smooth muscle alpha-actin (SMalphaA) gene, an important determinant of myofibroblast differentiation and early protein marker for stromal cell response to tissue injury. TGFbeta1 is a well known transcriptional activator of the SMalphaA gene in myofibroblasts. In contrast, thrombin independently elevates SMalphaA expression in human pulmonary myofibroblasts at the posttranscriptional level. A common feature of SMalphaA up-regulation mediated by thrombin and TGFbeta1 is the involvement of the cold shock domain protein YB-1, a potent repressor of SMalphaA gene transcription in human fibroblasts that also binds mRNA and regulates translational efficiency. YB-1 dissociates from SMalphaA enhancer DNA in the presence of TGFbeta1 or its Smad 2, 3, and 4 coregulatory mediators. Thrombin does not effect SMalphaA gene transcription but rather displaces YB-1 from SMalphaA exon 3 coding sequences previously shown to be required for mRNA translational silencing. The release of YB-1 from promoter DNA coupled with its ability to bind RNA and shuttle between the nucleus and cytoplasm is suggestive of a regulatory loop for coordinating SMalphaA gene output in human pulmonary myofibroblasts at both the transcriptional and translational levels. This loop may help restrict organ-destructive remodeling due to excessive myofibroblast differentiation.

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Figures

Figure 1.
Figure 1.
(A) Immunoblot depicting SMαA protein accumulation in hPFBs over a 48-h period after exposure to TGFβ1 (0-5.0 ng/ml). (B) Northern blot showing actin mRNA levels (nonmuscle β-actin and γ-actin and SMαA) over a 16-h period after treatment of quiescent hPFBs with either TGFβ1 (5 ng/ml) or thrombin (2 or 10 U/ml). RNA sample from mouse AKR-2B embryonic fibroblasts after treatment with TGFβ1 (5 ng/ml, 16 h) is shown in the lane marked “+” to serve as a positive control for TGFβ1 response and to denote positions of actin mRNAs. An ethidium bromide-stained agarose gel showing 28 and 18S rRNAs is shown at the bottom to illustrate the relative amount of total RNA applied to each lane. (C) Immunoblots showing SMαA and β-actin (βA) protein levels in thrombin-treated (Thr, 5 U/ml) hPFBs (left). Thrombin-induced accumulation of SMαA was inhibited by cycloheximide (+ cycl, 10 μg/ml) but not actinomycin D (+ act D, 5 μg/ml) relative to thrombin alone (contl) as shown on the right.
Figure 2.
Figure 2.
DNA-binding assay showing diminished binding of YB-1 to its cognate pyrimidine-rich, single-stranded DNA binding site in the SMαA MCAT enhancer within 60 min after exposure of hPFBs to TGFβ1 (5 ng/ml). Changes in the size of the YB-1 protein pool in unfractionated hPFB nuclei were relatively modest over the first 60 min of exposure to TGFβ1 (right lanes).
Figure 3.
Figure 3.
(A) Nonhuman primate COS7 fibroblasts were transfected with VSMP4 plus either an empty mammalian expression vector (pCDNA3), an expression plasmid encoding the MSY-1 mouse homologue of YB-1 (MSY1), equal molar amounts of three expression plasmids encoding Smad 2, Smad 3, and Smad 4 (Smads) or a combination of all four expression plasmids (MSY1 + Smads). CAT ELISA performed 48 h after transfection revealed that MSY1 inhibited constitutive VSMP4 expression, whereas Smads were capable of activating VSMP4 as well as partly neutralizing the inhibitory effect of MSY-1 overexpression on VSMP4 activity. (B) Immunoblots showing native YB-1 levels in the nuclear, DNA-bound, and cytosolic fractions of fibroblasts after transfection with equal molar amounts of Smad 2, Smad 3, and Smad 4 expression plasmids. YB-1 interaction with a single-strand oligonucleotide probe containing its cognate, pyrimidine-rich binding site (YB-1:ssDNA-bound) decreased substantially by 6 h after transfection. Over the 48-h observation period, nuclear levels of YB-1 diminished (YB-1:nucleus) but increased in the cytosol (YB-1:cytosol). Smad 2 and Smad 3 proteins accumulated in the nuclear fraction within 24 h after transfection (Smad 2,3:nucleus).
Figure 4.
Figure 4.
(A) ELISA depicting CAT protein in transfected hPFBs that accumulated during a 10-min exposure to vehicle (solid bar) or thrombin (5 U/ml, shaded bar). hPFBs were transfected 48 h before treatment with the P4/CE(3′) reporter construct that contains the coding element (CE) from exon 3 of SMαA mRNA previously shown to bind YB-1 and Pur proteins required for translational repression of CAT mRNA (Kelm et al., 1999b). (B) Binding of native YB-1, Purα, and Purβ in hPFB nuclear and cytosol extracts to the exon 3 RNA coding element (3CE). Thrombin (5 U/ml, 10-min exposure) reduced binding of cytosolic YB-1 and Pur proteins to the 3CE RNA probe while enhancing these interactions in the nuclear compartment.
Figure 5.
Figure 5.
Immunofluorescence microscopic evaluation of YB-1 and SMαA distribution in thrombin-activated hPFBs. Nuclear compartmentalization of YB-1 and deployment of SMαA microfilaments were evident within 5 min of exposure to 5 U/ml thrombin (Th) compared with vehicle-treated controls (C). Nuclei were identified in YB-1 antibody-treated hPFBs by staining with DAPI (PI). Magnification, 40× objective.
Figure 6.
Figure 6.
Immunoblots depicting SMαA levels in hPFBs in the presence of TGFβ1 or thrombin with or without their cognate inhibitors. Lanes 1 and 6, vehicle-treated controls; lane 2, TGFβ1 alone; lane 3, TGFβ1 + SB431542 kinase inhibitor; lane 4, thrombin alone; lane 5, thrombin + SB431542 kinase inhibitor; lane 7, TGFβ1 alone; lane 8, TGFβ1 + thrombin protease inhibitor; lane 9, thrombin alone; lane 10, thrombin + thrombin protease inhibitor. Each inhibitor attenuated SMαA expression in an independent, ligand-specific manner with little effect on GAPDH expression.
Figure 7.
Figure 7.
(A) Thrombin-dependent changes in YB-1 subcellular compartmentalization required the Ras-Raf-MEK-ERK mitogen-activated protein (MAP) kinase signaling pathway. Immunoblot analysis showed that YB-1 levels decreased in the cytosol and markedly increased in the nucleus of hPFBs within 1 min after treatment with thrombin (1 U/ml). The MAP kinase inhibitors U0126 and PD98059 both blocked thrombin-dependent nuclear import of YB-1. GAPDH expression in the two protein fractions indicated equivalent protein loading for each sample and was largely unaffected by the various treatments. The nomenclature 1/1′ and 1/5′ denotes 1 U/ml thrombin for either 1 or 5 min, respectively. (B) Enhanced SMαA synthesis in thrombin-treated hPFBs required ERK protein phosphorylation. Immunoblot analysis was performed using antibodies specific for SMαA and phosphorylated forms of p44 and p42 ERK signaling intermediaries. A 5-min exposure to thrombin caused rapid phosphorylation of p42 and p44 and enhanced SMαA synthesis but was without effect in hPFBs that were treated with the U0126 MEK1 inhibitor.
Figure 8.
Figure 8.
Hypothetical scheme for coordination of SMαA transcription and translation by YB-1 in TGFβ1- and thrombin-activated hPFBs. Rapid SMαA translation and thin filament deployment by thrombin may commence from an available pool of preexisting, cytosolic SMαA mRNPs. Replenishment and amplification of this pool may require de novo activation of the SMαA gene via a slower, TGFβ1-dependent transcriptional process requiring both nuclear uptake of Smads plus dissociation of YB-1 from the SMαA promoter. Displaced YB-1 may be sequestered from the nucleus within cytosolic SMαA mRNPs. As ERK-dependent translation of SMαA messenger RNA proceeds, YB-1 nuclear reentry may terminate TGFβ1-dependent transcription and myofibroblast differentiation.
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References

    1. Armstrong, A. T., Strauch, A. R., Starling, R. C., Sedmak, D. D., and Orosz, C. G. (1997). Morphometric analysis of neointimal formation in murine cardiac grafts. III. Dissociation of interstitial fibrosis from neointimal formation. Transplantation 64, 1198-1202. - PubMed
    1. Becker, N. A., Kelm, R. J., Jr., Vrana, J. A., Getz, M. J., and Maher, L.J.I. (2000). Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle α-actin promoter during myofibroblast differentiation. J. Biol. Chem. 275, 15384-15391. - PubMed
    1. Bogatkevich, G. S., Tourkina, E., Silver, R. M., and Ludwicka-Bradley, A. (2001). Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway. J. Biol. Chem. 276, 45184-45192. - PubMed
    1. Capowski, E. E., Esnault, S., Bhattacharya, S., and Malter, J. S. (2001). Y box-binding factor promotes eosinophil survival by stabilizing granulocyte-macrophage colony-stimulating factor mRNA. J. Immunol. 167, 5970-5976. - PubMed
    1. Carlini, L. E., Getz, M. J., Strauch, A. R., and Kelm, R. J., Jr. (2002). Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Purα, Purβ, and MSY1. J. Biol. Chem. 277, 8682-8692. - PubMed

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