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Vol. 16, Issue 10, 4931-4940, October 2005

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YB-1 Coordinates Vascular Smooth Muscle -Actin Gene Activation by Transforming Growth Factor 1 and Thrombin during Differentiation of Human Pulmonary Myofibroblasts

Aiwen Zhang ^*, Xiaoying Liu ^*, John G. Cogan ^*, Matthew D. Fuerst ^*, John A. Polikandriotis ^* , Robert J. Kelm, Jr. , and Arthur R. Strauch ^*

^* Department of Physiology and Cell Biology, The Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210; Department of Medicine, College of Medicine, University of Vermont, Colchester, VT 05446

Submitted March 16, 2005; Revised July 7, 2005; Accepted August 2, 2005
Monitoring Editor: Carl-Henrik Heldin

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
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 1 (TGF1) and thrombin, in directing expression of the vascular smooth muscle -actin (SMA) gene, an important determinant of myofibroblast differentiation and early protein marker for stromal cell response to tissue injury. TGF1 is a well known transcriptional activator of the SMA gene in myofibroblasts. In contrast, thrombin independently elevates SMA expression in human pulmonary myofibroblasts at the posttranscriptional level. A common feature of SMA up-regulation mediated by thrombin and TGF1 is the involvement of the cold shock domain protein YB-1, a potent repressor of SMA gene transcription in human fibroblasts that also binds mRNA and regulates translational efficiency. YB-1 dissociates from SMA enhancer DNA in the presence of TGF1 or its Smad 2, 3, and 4 coregulatory mediators. Thrombin does not effect SMA gene transcription but rather displaces YB-1 from SMA 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 SMA 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Fibrosis is a serious complication of chronic cardiopulmonary diseases and postsurgical complication of heart and lung transplant (Pickering and Boughner, 1990; Armstrong et al., 1997; Howell et al., 2002; Chapman, 2004). Although management of acute allograft rejection is accomplished through the use of immunosuppressive agents that can limit immune cell infiltration, the cause of chronic rejection and failure in accepted allografts is poorly understood thus offering no treatment solution short of retransplant. Leading the formation of scar tissue is a specialized stromal cell referred to as the myofibroblast that contains abundant microfilament networks composed of smooth muscle-specific contractile protein isoforms (Tomasek et al., 2002; Hinz and Gabbiani, 2003; Grotendorst et al., 2004). Chronic accumulation of myofibroblasts is associated with excessive extracellular matrix protein biosynthesis, hypercontractility, and organ-destructive remodeling. Vascular smooth muscle -actin (SMA) is one of the major contractile proteins expressed by differentiated myofibroblasts (Darby et al., 1990; Ronnov-Jessen and Petersen, 1996; Hinz et al., 2001; Cogan et al., 2002). Normally, SMA-enriched myofibroblasts are early, transient participants in stromal wound healing processes. We reason that characterization of the initial molecular events associated with activation of the SMA gene in stromal cells would help identify novel checkpoints for regulating the extent of myofibroblast involvement in wound healing. Therapeutic interventions aimed at managing potentially rate-limiting steps in myofibroblast differentiation may help protect against uncontrolled tissue remodeling and the so-called "endless healing" phenotype (Tomasek et al., 2002) with attendant hypertrophic scarring and loss of parenchymal cell function.

In recent studies from our laboratory, we have observed that the SMA gene is transcriptionally activated by the cytokine TGF1 via Smad-dependent signaling (Cogan et al., 2002; Subramanian et al., 2004). In the context of wound healing, this potent, profibrotic cytokine is released from a latent state at the site of tissue injury by activators intimately associated with local monocyte/macrophage infiltration and adhesion such as matrix metalloproteinase (MMP)-2 and -9, thrombospondin-1, and V6 integrin (Leask and Abraham, 2004). Less well understood, and especially relevant to the situation of organ transplantation in the immunosuppressed human patient population, is the role of nonimmune based surgical trauma and graft ischemic/reperfusion injury in promoting myofibroblast activation and chronic tissue remodeling. The serine protease thrombin, released during donor organ harvest and subsequent engraftment not only initiates the enzyme cascade responsible for coagulation but also has been reported to activate latent TGF1 (Howell et al., 2002) and possess profibrotic and extravascular cellular signal transduction properties that lead to tissue remodeling in both the native and transplanted heart and lung (Erlich et al., 2000; Holschermann et al., 2000; Mackman, 2003). In this regard, our studies of syngeneic murine heart grafts has revealed a form of alloantigen-independent organ remodeling (Armstrong et al., 1997) that evolves slowly but resembles in form the far more robust perivascular and interstitial fibrotic response observed in long-term cardiac allografts in immunosuppressed recipients (Subramanian et al., 2002). In accepted allografts, we hypothesize that persistent, low-grade immune cell infiltration amplifies an underlying peri-transplant wound healing response based on the profibrotic action of TGF1 and thrombin released as a consequence of mechanical and/or reperfusion injury during transplant surgery. Amplification of these nonimmune-based wound healing processes by alloantigen or alloantibody augments dysfunctional remodeling leading to premature graft failure.

To more fully understand the molecular mechanism of peri-transplant wound healing, we have examined the effect of thrombin on SMA gene regulation in human pulmonary fibroblasts (hPFBs) and compared its action to TGF1, a well known transcriptional activator of the smooth muscle actin gene. In contrast to regulatory mechanism reported for TGF1, thrombin had a significant, potentially supplemental effect on SMA translation. A common feature of SMA up-regulation mediated by both thrombin and TGF1 was reliance on the cold shock domain protein YB-1 (Matsumoto and Wolffe, 1998; Wolffe, 1998) that represses SMA gene transcription in stromal fibroblasts yet exhibits additional RNA binding properties that regulate mRNA translational efficiency. YB-1 dissociated from SMA enhancer DNA and was exported from the nucleus in the presence of TGF1 or its Smad coregulatory mediators. On the other hand, thrombin displaced YB-1 from SMA exon 3 mRNA coding sequences previously shown to be required for translational silencing (Kelm et al., 1999b). Thrombin-facilitated RNA release and nuclear reentry of YB-1 provides compelling evidence for a regulatory loop to control SMA gene output via coordinate control at both the transcriptional and translational levels. Published evidence indicates that thrombin activates several growth and inflammation-associated genes and that promoter regions flanking these genes contained binding sites for YB-1 (Minami et al., 2004). Besides reducing SMA gene expression, YB-1 also represses type I collagen 1 and 2 gene transcription (Norman et al., 2001; Higashi et al., 2003). Thus, nuclear YB-1 may temper TGF1-dependent, myofibroblast accumulation while stimulating inflammatory cytokines necessary for angiogenesis, restored microperfusion, and infiltration of organ-reconstructive circulating progenitor cells into the wound provisional matrix.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell Culture Methods and Preparation of Protein Extracts
Human pulmonary fibroblasts (hPFB) were established in primary culture from enzyme-dispersed tissue fragments of human neonatal lung tissue obtained at autopsy and were the kind gift of Dr. Daren L. Knoell (Departments of Pharmacy and Internal Medicine, The Ohio State University, Columbus, OH). Pulmonary fibroblasts were maintained in a 1:1 mixture of Ham's F-12 and DMEM (1.0 g/l D-glucose) supplemented with penicillin-streptomycin-Fungizone, Gentamicin (50 µg/ml), and 10% heat-inactivated fetal bovine serum (hiFBS; all culture medium reagents from Invitrogen, Carlsbad, CA). Nonhuman primate COS7 kidney fibroblasts were maintained in DMEM (4.5 g/l D-glucose) supplemented with penicillin-streptomycin and 10% hiFBS. Cell lines were cultivated in a humidified incubator at 37°C at 5% CO₂. Fibroblasts were rendered quiescent by a 48-h exposure to HEPES-buffered DMEM (1.0 g/l D-glucose) containing 0.5% hiFBS and penicillin-streptomycin-Fungizone. Recombinant human TGF1 (5 ng/ml, final concentration; R&D Systems, Minneapolis, MN) was added to cultures for varying periods before preparation of protein extracts. Human plasma thrombin (1000 NIH U/mg protein) was obtained from Calbiochem (La Jolla, CA) and used between 1 U and 10 U/ml as noted in the text. The metabolic inhibitors actinomycin D and cycloheximide were used at 5 and 10 µg/ml, respectively, in experiments on thrombin-activated myofibroblasts. Inhibitors of the TGF1 type I receptor serine/threonine kinase (SB431542; Sigma-Aldrich, St. Louis, MO) and thrombin serine protease (Thromstop, N--NAPAP; American Diagnostica, Stamford, CT) were administered at 1 and 0.1 µM, respectively, for 1 h before exposing hPFBs to medium supplemented with TGF1 or thrombin plus fresh aliquots of inhibitors. For protein extract preparations, cell monolayers were washed twice with Dulbecco's phosphate-buffered saline (PBS), scraped into fresh PBS, sedimented at 3000 rpm, washed once more in PBS, and resuspended in eight packed-cell volumes of hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], and 0.5 mM dithiothreitol [DTT]). Cells were allowed to swell for 10 min on ice before transfer to a Dounce homogenizer for processing with a type B pestle. Nuclei were collected from ruptured cells by centrifugation for 15 min at 4000 rpm and suspended in one-half packed-pellet volume of ice-cold, low salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 0.02 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT). High salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) equal to one-half packed-pellet volume was added, and the nuclei were further extracted with gentle rocking for 30 min at 4°C. Nuclei were collected by centrifugation for 30 min at 14,500 rpm and dialyzed against 50 volumes of dialysis buffer containing 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT. After dialysis, supernatants were collected by centrifugation at 14,500 rpm for 20 min for use in biochemical assays. Whole cell extracts were prepared from PBS-rinsed monolayers using radioimmunoprecipitation (RIPA) buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail, 0.2 mM PMSF, and 0.5 mM DTT) at 0.6 ml/100-mm culture plate followed by gentle rocking for 15 min at 4°C. Adherent lysed cell remnants were scraped into 0.3 ml of RIPA buffer and combined with the original lysate into a single microcentrifuge tube and the supernatant fraction was collected at 10,000 x g for 10 min at 4°C.

DNA- and RNA-binding Assays
Several synthetic biotinylated oligonucleotide probes were used in this study as follows: 1) a pyrimidine-rich, 30-base segment of single-strand DNA encompassing the reverse strand of the 5'-flanking region of the mouse SMA gene located between -195 and -164 (also referred to as D_R, 5'-ccctcgtcttgtctccttacgtcaccttctct-3') and previously shown to bind YB-1 with high affinity; Cogan et al., 1995; Kelm et al., 1999a); 2) the RNA coding element located in exon 3 of SMA mRNA (CE-RNA, 5'-gggaguaaugguuggaaugggccaaaaaga-3'), previously shown to bind YB-1 and Pur proteins (Kelm et al., 1999b); and 3) its mutant counterpart (CE-RNAmut, 5'-uugaguaaugguuuuccguggccaaccaga-3'). Underlined sequences denote regions changed by mutation. Reaction mixtures containing protein extract (100 µg of protein) and biotinylated oligonucleotides (100 pmol; Integrated DNA Technologies, Coralville, IA) were incubated in a buffer containing poly(dI-dC), 10 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 0.12 mM PMSF, 4% glycerol. Protein-biotinylated DNA complexes were captured on streptavidin-immobilized paramagnetic particles (Promega, Madison, WI; 0.6 ml/reaction, 30-min incubation) as described previously (Cogan et al., 2002; Subramanian et al., 2004). After washing four times with buffer containing 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 100 mM NaCl, bound protein was eluted using 2x protein denaturing buffer and analyzed by SDS-PAGE and immunoblotting procedures.

Mammalian Protein Overexpression Plasmids, Cell Transfection, and Reporter Gene Assays
Fibroblasts at 40-50% confluence were transfected with mixtures of the previously described SMA promoter:reporter fusion plasmids (VSMP4 and VSMP8) plus plasmids encoding various transcriptional regulatory proteins (Cogan et al., 2002; Subramanian et al., 2004). The Mirus (Invitrogen, San Diego, CA) transfection reagent was used with a protocol provided by the manufacturer. VSMP4 contains multiple SMA promoter regulatory elements including two different transcriptional activation elements referred to as SPUR (Subramanian et al., 2004) and THR (Cogan et al., 2002). VSMP8 has been widely used to generate transgenic mouse lines and contains a 3.6-kb 5'-flanking/first intron promoter fragment from mouse SMA genomic DNA containing both smooth muscle-specific developmental control and tissue injury response elements (Wang et al., 1997, 1998; Maeda et al., 1999; Lopes et al., 2003; Hassanain et al., 2005). For translation assays, the P4/CE (3') construct was made by incorporating the exon 3 coding sequence of the mouse SMA gene into VSMP4 as described previously (Kelm et al., 1999b). The exon 3 coding element previously was shown to repress chloramphenicol acetyl-transferase (CAT) reporter gene mRNA translation, but not transcription, when placed in the 5'-untranslated region of the CAT reporter gene at a position 3' to the transcriptional start site. Plasmids encoding the human Smad 2, 3, and 4 proteins were kindly provided by Drs. L. Choy and R. Derynck (University of California, San Francisco, San Francisco, CA). Plasmids were purified using QIAGEN preparative resin and a protocol provided by the manufacturer (QIAGEN, Valencia, CA). Forty-eight hours after transfection, cells were washed three times with ice-cold PBS and then lysed using CAT enzyme-linked immunosorbent assay (ELISA) lysis buffer (Roche Diagnostics, Indianapolis, IN). Whole cell extracts were clarified at 14,000 x g for 10 min at 4°C and briefly stored at -20°C before immunoassay. Total protein in extracts was determined by BCA colorimetric assay (Pierce Chemical, Rockford, IL), and reporter gene activity was determined using a commercial CAT ELISA kit (Roche Diagnostics) and expressed on a per microgram protein basis. Transfections were performed in triplicate and repeated three to five times. Data sets were subjected to analysis of variance to assess statistical significance set at p < 0.05.

View larger version (69K): [in this window] [in a new window]   Figure 1. (A) Immunoblot depicting SMA protein accumulation in hPFBs over a 48-h period after exposure to TGF1 (0-5.0 ng/ml). (B) Northern blot showing actin mRNA levels (nonmuscle -actin and -actin and SMA) over a 16-h period after treatment of quiescent hPFBs with either TGF1 (5 ng/ml) or thrombin (2 or 10 U/ml). RNA sample from mouse AKR-2B embryonic fibroblasts after treatment with TGF1 (5 ng/ml, 16 h) is shown in the lane marked "+" to serve as a positive control for TGF1 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 SMA and -actin (A) protein levels in thrombin-treated (Thr, 5 U/ml) hPFBs (left). Thrombin-induced accumulation of SMA 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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Differential Action of TGF1 and Thrombin on SMA Expression in Human Pulmonary Myofibroblasts
Thrombosis and fibrosis, mediated by the actions of thrombin and TGF1, respectively, are tissue repair processes that minimize blood loss and facilitate construction of a myofibroblast-enriched, provisional extracellular matrix needed to close open wounds. Of particular interest were possible similarities between TGF1- and thrombin-based SMA gene induction required for conversion of stromal fibroblasts into contractile smooth muscle-like myofibroblasts. As a first approach, we compared the effects of TGF1 and thrombin on the behavior of single strand-specific, nucleic acid-binding proteins first identified by our laboratories as essential negative regulators of SMA gene transcription in rodent myofibroblasts and smooth muscle cells (Cogan et al., 1995; Sun et al., 1995; Kelm et al., 1999a). SMA gene expression in primary cultures of hPFBs exhibited a robust, 9-fold increase in response to TGF1 over the same concentration range previously demonstrated to be effective for gene stimulation in mouse embryonic fibroblast and neonatal rat aortic smooth muscle cell lines (Figure 1A). TGF1 also stimulated more than a 10-fold increase in transcription from reporter genes containing a 191-base pair mouse SMA enhancer element harboring TGF1-activating elements (VSMP4) as well as a larger 3.6-kb 5'-flanking/first intron promoter fragment (VSMP8) from mouse SMA genomic DNA (Min et al., 1990) that contains additional smooth muscle-specific developmental control elements (our unpublished data). Similarly, transcription of SMA mRNA from the native human SMA gene was markedly enhanced in hPFBs by TGF1 administered over a 16-h period (Figure 1B). In contrast, Northern blots prepared from hPFBs exposed to either 2 or 10 U/ml thrombin showed only a very modest increase in nonmuscle - and -actin mRNA levels between 2 and 8 h before settling back to a low baseline level of expression after 16 h (Figure 1B). Despite its rather unremarkable effect on net SMA mRNA accumulation, however, thrombin administered to hPFB at doses between 2-10 U/ml caused a rapid and significant increase in SMA protein level (Figure 1C). Evaluation of Western blots prepared from hPFBs treated with 5 U/ml thrombin plus the metabolic inhibitors actinomycin D or cycloheximide indicated a requirement for active SMA protein biosynthesis rather than de novo SMA mRNA transcription (Figure 1C). Together, the data indicated that rapidly elevated SMA protein synthesis in hPFBs after exposure to thrombin seemed to be based largely on a posttranscriptional control mechanism involving highly efficient utilization of a small pool of available SMA mRNA.

View larger version (24K): [in this window] [in a new window]   Figure 2. DNA-binding assay showing diminished binding of YB-1 to its cognate pyrimidine-rich, single-stranded DNA binding site in the SMA MCAT enhancer within 60 min after exposure of hPFBs to TGF1 (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 TGF1 (right lanes).

View larger version (39K): [in this window] [in a new window]   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).

TGF1 Dissociates YB-1 Repressor from the Activated SMA Promoter

Reduced interaction between YB-1 protein and SMA enhancer DNA in TGF1-activated human myofibroblasts supported our previous findings showing that cold-shock domain proteins function to repress SMA gene transcription (Kelm et al., 1999a; Carlini et al., 2002). However, our previous studies on mouse embryonic fibroblasts containing the MSY-1 homologue of human YB-1 provided only indirect evidence for repressor function because the experimental approach was designed simply to reveal a general increase in transcriptional activity when point mutations known to block MSY-1 binding to Pyr-rich, ssDNA were placed into the context of SMA enhancer:reporter fusion genes (Cogan et al., 1995; Sun et al., 1995). The binding of other SMA gene repressors, Pur and Pur, also was inhibited in these mutant constructs thus potentially obscuring the specific contribution of MSY-1 to gene repression. To examine the regulatory properties of MSY-1 more directly, nonhuman primate COS7 kidney fibroblasts were transfected with the TGF1-responsive mouse SMA enhancer construct VSMP4 plus a mammalian expression plasmid encoding full-length mouse MSY-1 cDNA. As shown in Figure 3A, overexpression of mouse MSY-1 protein in simian COS7 fibroblasts was sufficient to block baseline transcription from the VSMP4 enhancer consistent with its action as a SMA gene repressor. Additional results presented in Figure 3A indicate that combined overexpression of Smad proteins 2, 3, and 4 caused more than a twofold increase in SMA enhancer activity in COS7 fibroblasts as well as significantly offset the inhibitory effect of mouse MSY-1 protein overexpression on enhancer transcriptional activity. These observations were an important extension of our recent studies showing that Smads also can partly relieve repression of the SMA enhancer by the Pur protein (Subramanian et al., 2004). To examine how Smad protein overexpression influenced the distribution and DNA-binding properties of endogenous YB-1 protein, we examined nuclear, cytosolic, and DNA-bound protein fractions by Western blot analysis. Activation of the SMA enhancer by overexpression of Smads 2, 3, and 4 during a 48-h period was accompanied by a decrease in the level of YB-1 protein in COS7 nuclei with concomitant accumulation in the cytoplasmic fraction (Figure 3B). Depletion of YB-1 from the nucleus also was accompanied by decreased interaction with its cognate, Pyr-rich ssDNA enhancer-binding element. Interestingly, ssDNA-YB-1 interaction decreased rapidly subsequent to Smad protein overexpression and was substantially reduced in parallel with depletion of YB-1 from the nucleus. Nuclear uptake of Smads 2 and 3 exhibited a notable lag phase and was not significantly increased until 24-48 h after transfection (Figure 3B) when a significant increase in VSMP4 enhancer activity was noted (our unpublished data). The results imply that interruption of YB-1 interaction with single-strand SMA enhancer DNA and nuclear export were early responses to Smad protein overexpression in transfected fibroblasts.

View larger version (28K): [in this window] [in a new window]   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 SMA 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, Pur, and Pur 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.

Thrombin Disrupts Interaction of YB-1 and the Pur / Corepressor Proteins with Cytoplasmic SMA mRNA

Rapid Deployment of SMA Thin Filaments and Nuclear Uptake of YB-1 in Myofibroblasts Is Mediated by Thrombin-specific Signaling
Net redistribution of YB-1 from the cytosol to the nucleus occurred rapidly in thrombin-treated hPFBs. Immunofluorescence microscopy was used to examine spatial relationships between the subcellular distribution of YB-1 and assembly of SMA thin filaments in thrombin-treated hPFBs. Within 5 min after exposure to thrombin, diffusely distributed cytosolic YB-1 was consolidated within myofibroblast nuclei (Figure 5). Brief exposure to thrombin also resulted in a striking increase in deployment of SMA thin filament networks in hPFBs. Together with the results of biochemical fractionation and SMA mRNA binding studies shown in Figure 4, the morphological analysis of thrombin-activated myofibroblasts implied that relocalization of YB-1 from the cytosol to nuclear compartment was temporally linked to rapid assembly of the SMA cytoskeleton in myofibroblasts. Because thrombin has been implicated as one of the several cellular protease capable of activating latent TGF1 stores in stromal cells, we performed additional studies to evaluate the relative contribution of TGF1- and thrombin-specific receptor signaling in myofibroblast SMA protein accumulation. As shown in Figure 6, inclusion of an inhibitor of the type I TGF1 receptor serine/threonine kinase in the culture medium (SB431542) substantially reduced SMA protein accumulation in TGF1-treated hPFBs, but it had no grossly restrictive effect on induction by thrombin. Further supporting an independent molecular mechanism of action, a thrombin serine protease inhibitor attenuated SMA accumulation in thrombin-treated hPFBs but did not prevent induction in the presence of TGF1 (Figure 6). The ability of thrombin to rapidly augment SMA expression in hPFBs does not seem to involve amplification of an underlying TGF1-dependent process such as activation of latent TGF1 or increased mRNA transcription but instead seems to have its basis in a thrombin-specific action such as enhanced efficiency of SMA protein synthesis and/or thin filament stability. In this regard, phosphorylated extracellular signal-regulated kinase (ERK) proteins have been shown to mediate thrombin PAR-1 receptor-based signal transduction and promote rapid elevation of smooth muscle-specific myosin gene expression in thrombin-treated vascular smooth muscle cells (Reusch et al., 2001). As shown in Figure 7A, the Ras-Raf-(mitogen-activated protein kinase kinase (MEK)-ERK pathway inhibitors U0126 and PD98059 both blocked nuclear uptake of YB-1 and increased the size of the cytosolic YB-1 pool relative to baseline levels seen in control hPFB preparations. MEK1 inhibition by U0126 also partly blocked accumulation of SMA protein in thrombin-treated hPFBs while causing a notable reduction in the level of phosphorylated p44/p42 ERK signaling intermediary proteins in hPFBs (Figure 7B).

View larger version (78K): [in this window] [in a new window]   Figure 5. Immunofluorescence microscopic evaluation of YB-1 and SMA distribution in thrombin-activated hPFBs. Nuclear compartmentalization of YB-1 and deployment of SMA 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, 40x objective.

View larger version (69K): [in this window] [in a new window]   Figure 6. Immunoblots depicting SMA levels in hPFBs in the presence of TGF1 or thrombin with or without their cognate inhibitors. Lanes 1 and 6, vehicle-treated controls; lane 2, TGF1 alone; lane 3, TGF1 + SB431542 kinase inhibitor; lane 4, thrombin alone; lane 5, thrombin + SB431542 kinase inhibitor; lane 7, TGF1 alone; lane 8, TGF1 + thrombin protease inhibitor; lane 9, thrombin alone; lane 10, thrombin + thrombin protease inhibitor. Each inhibitor attenuated SMA expression in an independent, ligand-specific manner with little effect on GAPDH expression.

View larger version (48K): [in this window] [in a new window]   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 SMA synthesis in thrombin-treated hPFBs required ERK protein phosphorylation. Immunoblot analysis was performed using antibodies specific for SMA 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 SMA synthesis but was without effect in hPFBs that were treated with the U0126 MEK1 inhibitor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
In this report, we described a novel role for the YB-1 nucleic acid-binding protein in coordinating SMA gene expression in human pulmonary myofibroblasts. YB-1 contains a highly conserved cold-shock domain that mediates nucleic acid binding in response to cellular stress signals, including low temperature, drug toxicity, reactive oxygen signaling, and UV damage (Matsumoto and Wolffe, 1998) (Kohno et al., 2003). In addition to SMA, several other genes important in wound healing and cellular proliferation and survival also are regulated by YB-1, including those encoding 1 and 2 (I) collagen (Norman et al., 2001; Higashi et al., 2003), the B chain isoform of platelet-derived growth factor (PDGF) (Steinina et al., 2000), MMP-2 (Mertens et al., 1997), vascular endothelial growth factor (Coles et al., 2004), granulocyte macrophage-colony stimulating factor (Coles et al., 2000), p21 and p53 (Kohno et al., 2003), and the Fas death receptor (Lasham et al., 2000). In our recent studies of myofibroblasts and arterial smooth muscle cells, we discovered that MSY-1, the YB-1 homologue in rodents, was able to repress SMA gene transcription by binding the pyrimidine-rich strand of the MCAT enhancer located in an inverted repeat between positions -190 and -150 of the SMA promoter (Kelm et al., 1999a; Carlini et al., 2002). Alternative base pairing within the inverted repeat produces a thermodynamically stable stem-loop structure. The pyrimidine-rich, reverse-strand component of the loop binds YB-1 thus potentially disrupting promoter interaction with transcriptional activators such as Smad 2, Smad 3, Sp1, and TEF-1 that require duplex DNA proximal to the MCAT enhancer (Cogan et al., 2002; Subramanian et al., 2004). The MCAT enhancer region also exhibits substantial chromatin conformational change in response to TGF1 (Becker et al., 2000). Relationships between YB-1, RNA binding, and thrombin signaling also were noted and of interest because thrombin activation of PDGF-B gene transcription in human endothelial cells has been shown to require YB-1 proteolytic processing and nuclear translocation (Steinina et al., 2000).

Results reported here extend findings of TGF1-regulated interplay between Sp1, a GC-rich SMA promoter element called SPUR, and the Pur family of single-strand nucleic acid binding proteins in myofibroblasts (Subramanian et al., 2004). Although YB-1 also exhibits TGF1-dependent interplay with the SMA promoter, its action is centered on the MCAT enhancer located 120 base pairs upstream from SPUR. However, the ability of YB-1 and Pur repressor proteins to self-associate and bind diverse transcriptional activators, including Sp1, serum response factor, TEF-1, and Smad proteins (Kelm et al., 1999a, 2003; Subramanian et al., 2004) implies that they do not function in complete physical isolation. Although originally identified as repressors of SMA gene transcription, YB-1 and Pur proteins also have roles in RNA processing and transport of RNPs along cytoskeletal filaments in the cytoplasm (Ruzanov et al., 1999) (Ohashi et al., 2000, 2002; Stickeler et al., 2001; Kohno et al., 2003; Kanai et al., 2004). The YB-1 protein family member p50 is one of the most abundant components of ribonucleoprotein particles in eukaryotes and has a notable mRNA-dependent affinity for muscle and nonmuscle actin filaments (Ruzanov et al., 1999). Excessive amounts of p50 in RNPs effectively blocked both translation and F-actin interaction, whereas high mRNA:p50 ratios were associated with active polyribosomal RNPs and RNP-actin filament complexes. Titration studies using human recombinant YB-1 and atomic force microscopy revealed that RNPs with a low YB-1: mRNA ratio were unfolded and better configured for efficient polyribosomal binding and translation (Skabkin et al., 2004). Published reports also indicated that C-terminal proteolytic processing (Steinina et al., 2000) may influence YB-1 shuttling dynamics between the nuclear and cytoplasmic compartments thus providing another control point for governing protein function. Pur proteins also have been identified in RNPs where they form links with motor proteins such as kinesin and myosin Va and IIB in neuronal cells (Ohashi et al., 2000, 2002; Kanai et al., 2004). Pur protein-RNPs are thought to couple microtubules with kinesin motor proteins to transport mRNAs from their sites of synthesis in cell bodies to distal polyribosomes located near synaptic terminals.

As RNA-binding proteins, YB-1 and Pur proteins perform unique tasks in the cytosol that complements their more conventional roles as nuclear transcriptional regulatory proteins. In this regard, myofibroblasts rapidly assemble a polarized SMA thin filament system capable of producing directional forces necessary for reshaping the provisional extracellular matrix and guiding wound contraction. We speculate that myofibroblasts use YB-1 and Pur proteins to coordinate the transcription, transport, and compartmentalization of SMA mRNA in a manner that facilitates efficient construction and deployment of a polarized smooth muscle-like contractile apparatus at sites of tissue injury. In this scheme, nuclear YB-1 and Pur proteins that become released from SMA enhancer DNA in TGF1-activated myofibroblasts may no longer function as transcriptional repressors but instead assist as chaperones in the packaging, transport, and translation of SMA mRNA. RNA-based actions of YB-1 and Pur proteins seem to be directed by thrombin signaling that likely coexists with TGF1 signaling within the complex microenvironment of the healing wound. Transience is a key feature of SMA gene expression and thin filament deployment in myofibroblasts and we speculate that thrombin-directed release of YB-1 and Pur proteins from cytosolic RNPs and subsequent nuclear reentry may serve this requirement by rerepressing the SMA gene and terminating myofibroblast differentiation. Although requiring additional experiments, this hypothetical scheme constitutes a regulatory loop whereby SMA gene output in TGF1-activated myofibroblasts initially is amplified at the translational control level, then quenched by thrombin (Figure 8). The magnitude of SMA protein output in myofibroblasts subsequent to transcriptional activation by TGF1 may be rate-limited by thrombin-dependent shuttling of YB-1 and Pur proteins off cytosolic mRNA and onto enhancer DNA in the nucleus. The Ras-Raf-MEK-ERK signaling pathway stimulated by thrombin may be especially relevant in attenuation of TGF1 signaling because ERK-mediated phosphorylation of the Smad 2 or 3 linker region has been shown to prevent nuclear accumulation of these proteins (Kretzschmar et al., 1999). ERK signaling in pulmonary myofibroblasts might also influence ribosomal protein phosphorylation and increase the net rate of protein synthesis during wound healing similar to its role in mechanical force-injured cardiac fibroblasts (MacKenna et al., 1998). On the other hand, thrombin may employ ERK to counteract TGF1 action on myofibroblast differentiation via induction of the Egr-1 DNA-binding protein known to activate promoters for proinflammatory tumor necrosis factor- (Verrecchia and Mauviel, 2004) and interleukin-1 (Tan et al., 2003) that both have well characterized anti-fibrotic, matrix-destructive properties. Related studies on endothelial cells showed that thrombin in some cases can counteract TGF1 signals because a PAR-1 peptide agonist caused TGF1 receptor internalization and suppressed Smad 2 and 3 nuclear translocation (Tang et al., 2005). Together, the available evidence suggests that thrombin may provide an important physiological rheostat designed to promote rapid construction of the contractile provisional matrix in healing wounds in parallel with hemostasis while preventing maladaptive tissue fibrosis due to chronically unchecked TGF1 signaling. A key question to be addressed by future experimentation is to determine whether the thrombin-mediated nuclear reentry of YB-1 and Pur proteins amplifies myofibroblast TGF1-dependent differentiation by facilitating the transport and translation of SMA mRNA, terminates the TGF1 signal by enabling YB-1 and Pur proteins to reaccess their cognate repressor sites in the SMA enhancer, or both. Chronic diseases associated with excessive accumulation of myofibroblasts, faulty SMA gene expression, and hypertrophic scarring such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, kidney epithelial mesenchymal transition, and transplant arteriosclerosis all may exhibit dysregulation in the coordination of thrombin and TGF1 signaling as a common predisposing condition.

View larger version (40K): [in this window] [in a new window]   Figure 8. Hypothetical scheme for coordination of SMA transcription and translation by YB-1 in TGF1- and thrombin-activated hPFBs. Rapid SMA translation and thin filament deployment by thrombin may commence from an available pool of preexisting, cytosolic SMA mRNPs. Replenishment and amplification of this pool may require de novo activation of the SMA gene via a slower, TGF1-dependent transcriptional process requiring both nuclear uptake of Smads plus dissociation of YB-1 from the SMA promoter. Displaced YB-1 may be sequestered from the nucleus within cytosolic SMA mRNPs. As ERK-dependent translation of SMA messenger RNA proceeds, YB-1 nuclear reentry may terminate TGF1-dependent transcription and myofibroblast differentiation.

Although the mechanistic details will require further study, disruption of YB-1 binding to the MCAT enhancer in the SMA promoter seems to be an early response to TGF1 treatments. SMA gene activation required Smad binding to multiple CAGA sites distributed within the large 40-base pair segment of DNA that contains the seven-base pair MCAT enhancer (Subramanian et al., 2004). There are three CAGA motifs between -190 and -150, but only two base pairs of one CAGA overlaps with the MCAT site. Thus, although loss of YB-1 binding could improve Smad access to one of its cognate sites in the promoter, the positions of the other two sites may render them less susceptible to YB-1 interference. Indeed, Smad binding is observed in a different region of the -190/-150 segment called the THR (TGF1 hyperreactivity region) but not at the MCAT where YB-1 binds (Subramanian et al., 2004). Chromatin conformation analysis before and after TGF1 also revealed that the MCAT-THR region unfolds during myofibroblast differentiation (Becker et al., 2000). Although the molecular basis for unfolding is not known, competitive interplay between YB-1 and Smads does not necessarily have to occur on promoter DNA but instead may involve protein complexes assembled in the cytosol that indirectly influence protein subunit availability or function in the nucleus. Data presented in Figure 3 imply that changes in YB-1 interaction with promoter DNA as well as the reduction in nuclear level of YB-1 both precede nuclear accumulation of Smad 2 and 3. In this regard, there is evidence that the anti-TGF1 properties of interferon- derive in part from its ability to foster binding of YB-1 to Smad 3 (Higashi et al., 2003). Within the complex, YB-1 prevents Smad 3 from linking with its p300 histone acetyl-transferase coactivator thus blocking transcription of the type 2(I) collagen gene in fibroblasts. Understanding dynamic interplay between YB-1, Smads, and MCAT-THR DNA will require more detailed examination of protein complex formation in the cytosol as well as protein-DNA interaction and assessment of DNA secondary structure in the nucleus. SMA gene activation may involve more sophisticated control than can be provided by simple replacements between repressors and activators on promoter DNA.

In summary, YB-1 represses SMA gene transcription and binds the MCAT region of the SMA promoter previously shown to undergo chromatin unfolding in TGF1-activated myofibroblasts. Transcriptional activation of the SMA gene by TGF1 was accompanied by loss of YB-1 binding to the MCAT enhancer and nuclear export. In contrast, thrombin stimulated SMA gene activation at the translational level and caused YB-1 and Pur proteins to dissociate from translational repression sites on mRNA in parallel with rapid SMA protein synthesis. Because thrombin by itself had no significant activating effect on SMA mRNA transcription, it will be important in future studies to establish whether thrombin-dependent nuclear reentry of YB-1 is designed to rerepress the SMA gene and/or destabilize preexisting SMA mRNA thus contributing to termination of both the TGF1 activation signal and myofibroblast differentiation. In a normally healing wound, myofibroblasts are transient participants and active forms of TGF1 and thrombin are likely to coexist. The ability of TGF1 and thrombin to independently exploit the DNA- and RNA-binding properties of YB-1, respectively, adds a new dynamic perspective to control of gene expression during myofibroblast differentiation that may reveal strategies for therapeutic management of these cells in chronic fibrotic diseases that affect the heart, lung, liver, and kidney.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grants R01 HL 60876 and P01 HL 70294 (to A.R.S.) and and R01 HL 54281 (to R.J.K.) and predoctoral fellowship 02151142B (to J.A.P.) from the American Heart Association.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-03-0216) on August 10, 2005.

Present address: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, Emory University, Atlanta, GA 30322.

Address correspondence to: Arthur R. Strauch (strauch.1{at}osu.edu).

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