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Published ahead of print on May 30, 2003, doi:10.1165/rcmb.2002-0315OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 583-590, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0315OC

Involvement of Serum Response Factor Isoforms in Myofibroblast Differentiation During Bleomycin-Induced Lung Injury

Yan Yang*, Xiaoning Zhe*, Sem H. Phan, Matt Ullenbruch and Lucia Schuger

Department of Pathology, Wayne State University School of Medicine, Detroit; and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan

Address correspondence to: Lucia Schuger, M.D., Department of Pathology, Wayne State University, 540 E. Canfield St., Rm. 9248, Detroit, MI 48201. E-mail: lschuger{at}med.wayne.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serum response factor (SRF) is a transcription factor essential for smooth muscle (SM) myogenesis. Its role in myofibroblast differentiation is, however, unknown. We studied the expression and the localization of SRF in bleomycin-induced pulmonary fibrosis, where myofibroblasts are abundant. We found that SRF levels were upregulated in bleomycin-exposed mouse lungs mainly due to de novo synthesis of SRF{Delta}5, a less myogenic SRF isoform. Before myofibroblast differentiation, SRF/SRF{Delta}5 was immunolocalized mostly in the cytoplasm of scattered fibroblasts at lesion sites. With the development of myofibroblasts, however, SRF/SRF{Delta}5 was found in myofibroblast nuclei. cDNA array analysis showed that SRF{Delta}5 and SRF induced expression of transforming growth factor-ß1, a critical factor in myofibroblast differentiation. This was accompanied by de novo expression of several inflammatory cell-specific mRNAs. The latter was confirmed by reverse transcriptase–polymerase chain reaction. Treatment of lung fibroblasts with tumor necrosis factor-{alpha}, which is produced early in the bleomycin model, induced SRF{Delta}5 expression and SRF/SRF{Delta}5 cytoplasmic accumulation, whereas addition of transforming growth factor-ß1 caused SRF/SRF{Delta}5 nuclear translocation followed by SM {alpha}-actin synthesis. Interleukin-4, another cytokine involved in myofibroblast differentiation, did not affect SRF or induce SRF{Delta}5 expression. Our studies therefore suggested a new mechanism whereby SRF and SRF{Delta}5 contribute to the emergence of myofibroblasts in lung injury and fibrosis.

Abbreviations: Dulbecco's Modified Eagle Media, DMEM • extracellular matrix, ECM • fetal bovine serum, FBS • interleukin, IL • matrix metalloproteinases, MMPs • plasma-derived sera, PDS • p-selectin glycoprotein ligand-1, PSGL-1 • reverse transcriptase–polymerase chain reaction, RT-PCR • serum response factor, SRF • smooth muscle, SM • tissue inhibitor of metalloproteinases, TIMPs • transforming growth factor-ß, TGF-ß • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Serum response factor (SRF) is a transcription factor with a critical role in smooth muscle (SM) myogenesis (1–3). SRF binds to the CArG box or CArG box-like motif, a cis-element present in multiple SM-specific proteins such as SM {alpha}-actin, SM22, SM myosin, ß-tropomyosin, caldesmon, etc., and stimulates their transcription (1–3). It has been previously shown that SRF has three different isoforms (SRF{Delta}5, SRF{Delta}45, and SRF{Delta}345) produced by alternative splicing (4, 5). These isoforms are expressed by some mature SM cells and increase or inhibit SM gene expression depending on the isoform size (5). In undifferentiated embryonic cells, however, only full SRF and SRF{Delta}5 are expressed (4, 6). In these cells SRF{Delta}5 behaves as a dominant-negative inhibitor (4, 6) due to its ability to interfere with SRF transactivation (4, 5). Moreover, SRF and SRF{Delta}5 are present in the nucleus, but mainly in the cytoplasm of undifferentiated embryonic mesenchymal cells. With the onset of myogenesis cytoplasmic SRF translocates to the nucleus, whereas SRF{Delta}5 nuclear/cytoplasmic distribution remains initially unchanged (7). Therefore, presence, intracellular distribution, and biological activity of SRF{Delta}5 seem to vary depending on the type of muscle cell and degree of maturation.

To determine whether SRF changes may occur in pathologic settings, we studied the expression and the localization of SRF in bleomycin-induced pulmonary fibrosis. The bleomycin-induced model of lung damage has been extensively applied to understand the pathophysiology of lung fibrosis (8–11). Active lung fibrosis is characterized by the presence of myofibroblasts, cells with a phenotype intermediate between SM and fibroblast (8, 12). All myofibroblasts express SM {alpha}-actin but not other SM-related proteins, although occasionally they have been reported to express one or two additional SM markers, depending on the type of myofibroblasts (12). Myofibroblasts play a main role in pulmonary fibrosis and other chronic fibrotic processes (8, 13–15). Relevant to the biology of myofibroblasts is their production of transforming growth factor (TGF)-ß1 and their sensitivity to this growth factor in terms of SM {alpha}-actin, collagen, and proteolytic enzymatic production (8, 11, 12).

After endotracheal administration of bleomycin, the murine lungs undergo an early inflammatory response (4–7 d) with focal appearance of myofibroblasts synthesizing collagens, fibronectin, and other extracellular matrix (ECM) components, resulting in fibrosis (10–21 d) (10, 11). Myofibroblast apoptosis and complete resolution of the fibrotic lesions occur if no additional bleomycin is administered (11, 16, 17). Among the main cytokines involved in bleomycin-induced damage are tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, and TGF-ß1. TNF-{alpha} and IL-1ß are detected in bleomycin-treated lungs early, before TGF-ß1 levels begin to increase and reach a peak around Days 7–10 after bleomycin instillation (10, 11, 18). Around the same time, TGF-ß1 reaches a plateau and slowly decreases along with the disappearance of myofibroblasts and lesion resolution (10, 11, 18).

The current studies showed that bleomycin instillation induced de novo synthesis of SRF{Delta}5 followed by upregulation of SRF. Early during bleomycin damage, SRF/SRF{Delta}5 immunoreactivity was enriched in the cytoplasm of scattered mesenchymal cells present at lesion sites. However, with the appearance of myofibroblasts, SRF/SRF{Delta}5 immunoreactivity became restricted to their nuclei. Transient transfections of adult mouse lung fibroblasts indicated that SRF{Delta}5 and SRF induced expression of many TGF-ß family members along with an unexpected expression of inflammatory cell markers. Exposure of these same cells to TNF-{alpha} and IL-1ß resulted in induction of SRF{Delta}5 and its enrichment in the cytoplasm, with no effect on SM {alpha}-actin expression. Treatment with TGF-ß1 caused SRF/SRF{Delta}5 nuclear translocation with upregulation of SM {alpha}-actin synthesis. IL-4, another soluble factor that stimulates SM {alpha}-actin synthesis (19), did not affect SRF/SRF{Delta}5. Furthermore, the combination of TNF-{alpha} or IL-1ß with TGF-ß1 resulted in all the changes seen in vivo, namely SRF{Delta}5 induction, SRF upregulation, nuclear translocation of these isoforms, and marked stimulation of SM {alpha}-actin synthesis. Our studies therefore suggested a novel mechanism contributing to the de novo emergence of the myofibroblast in bleomycin-induced lung injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bleomycin Model
Pulmonary fibrosis was induced in adult female CBA/J mice weighing 20–25 g (Jackson Labs, Bar Harbor, ME) by endotracheal injection of 1 U/kg body weight of bleomycin (Blenoxane; Bristol-Myers, Evansville, IN) in 30 µl of sterile saline under general anesthesia as previously described (20). Control animals received sterile saline only. Animals (six per group, two groups: bleomycin and control) were killed on different days (Days 1, 3, 7, 14, 17, and 21), and the lungs were used for these studies.

Immunohistochemistry
Five-micrometer-thick sections from formalin-fixed bleomycin-exposed lungs and matched controls were immunostained with antibodies against SRF, which also recognize SRF{Delta}5 (Santa Cruz Biotechnology, Santa Cruz, CA). In some experiments, bleomycin-treated lungs and their controls were additionally co-immunostained with antibodies against SM {alpha}-actin (Boehringer Mannheim Biochemical, Indianapolis, IN) and with antibodies against TGF-ß1 (R&D Systems Inc., Minneapolis MN). In addition, consecutive sections were immunostained for SM {alpha}-actin, desmin (DAKO Corp., Carpinteria, CA) and SM-myosin antibodies (Biomedical Technologies, Stoughton, MA). Adult lung fibroblasts were fixed with 100% ethanol for 10 min at room temperature. Antibodies to SM {alpha}-actin, SRF/SRF{Delta}5, and TGF-ß1 were used at a dilution of 1:50. Staining was completed using a commercial peroxidase–antiperoxidase kit (ABC Kit from Vector, Burlington, CA) or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Sigma, St. Louis, MO) as previously described (6, 21). Co-immunostaining for SRF/SRF{Delta}5 and SM {alpha}-actin was done according to a previous protocol (22).

Lung Fibroblasts Isolation
Adult murine lungs were minced in 0.1% collagenase I (Sigma) and incubated at 37°C for 60 min. Single cell suspensions were obtained, and fibroblasts were isolated by differential plating (23). The fibroblasts were then cultured in complete medium composed of DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/ml of penicillin (Sigma), 100 µg/ml streptomycin (Sigma), and 0.25 µg/ml of fungizone (Sigma). Because FBS may contain small amounts of TGF-ß1, in some experiments, the regular FBS was replaced by plasma-derived sera (PDS) from bovine stock (Cocalico Biologicals, Inc., Reamstown, PA), which contains no TGF-ß1. Contamination of cultures by epithelial cells, endothelial cells, or macrophages was ruled out before experimental use by performing reverse transcriptase–polymerase chain reaction (RT-PCR) for cytokeratin, PECAM, and CD68, respectively (not shown). A small number of SM {alpha}-actin positive cells, between 1 and 8%, was usually present in the cultures as indicated by SM {alpha}-actin immunostaining (not shown).

Immunoblot Analysis
Mouse adult lungs and lung fibroblasts were lysed and immunoblots were performed as previously described (24) using SM {alpha}-actin antibodies (Boehringer Mannheim) at a concentration of 0.25 mg/ml and SRF/SRF{Delta}5 antibodies (Santa Cruz) at a 1:200 dilution. Primary antibodies were detected with horseradish peroxidase–conjugated secondary antibodies diluted 1:3,000 (Bio-Rad Laboratories, Hercules, CA). The bands were visualized by chemiluminescence using a commercial kit (Amersham Life Sciences, Arlington Heights, IL) according to the manufacturer's instructions.

Construction of SRF- and SRF{Delta}5-Expressing Plasmids
SRF and SRF{Delta}5 cDNAs containing the full-length coding sequences, previously cloned into pGEM-T easy vector (Promega, Madison, WI) (5) were retrieved by EcoRI and ligated into EcoRI-digested pcDNA3 expression vector (Invitrogen). The orientation of the clones was determined by restriction digestion with PstI and the sequences were confirmed by nucleotide sequencing. These constructs were used for transfection into mouse lung fibroblasts and for array analysis.

Transient Transfections
Cells were grown in 6-well plates to 70% confluence and transfected using lipofectamine plus reagent (Invitrogen) following the manufacturer's instructions. The recombinant plasmids and null vector (used as control) were mixed with the transfection reagent in a 1:3.5 wt/vol proportion, and the cells were transfected for 3 h in OPTI-MEM I medium (Invitrogen). After 3 h of incubation at 37°C, the transfection medium was replaced with complete DMEM and the cultures were incubated for 18–24 additional hours. The plasmid constructs did not allow for determination of transfection efficiency because they expressed untagged protein. However, studies using GFP-tagged SRF plasmid constructs indicated that between 15 and 60% of the cells in our primary cultures were transfected depending upon each individual transfection (25).

cDNA Array Analysis
This was done using the Atlas cDNA Expression Array System from Clontech Laboratories Inc. (Palo Alto, CA) and following the manufacturer's instructions. Briefly, 5 µg of DNase I (Invitrogen)-treated total RNA was isolated from SRF-, SRF{Delta}5-, and null vector–transfected mouse lung fibroblasts using TRIzol (Invitrogen). Total RNA was incubated with random primers provided with the cDNA array kit at 50°C. Reaction buffer, dNTPs, 35 µCi {alpha}-32P-dATP (Amersham), and MMLV reverse transcriptase (Invitrogen) were then added and incubated for 25 min to allow cDNA extension. Reactions were terminated and purified by column chromatography. An aliquot of the eluted cDNAs was used to determine {alpha}-32P incorporation by scintillation counting. Equal counts of labeled probes from control, SRF{Delta}5- and SRF-transfected cell samples (> 100,000 counts/min) were added respectively onto nylon membranes containing over five hundred dotted cDNAs together with several housekeeping cDNAs as positive controls (Clontech, Cat.# 7741–1). The arrays were exposed to X-ray film (Kodak, Rochester, NY) for 18–72 h in the presence of an intensifying screen at –70°C. Exposure time was adjusted for each array until the signals for the housekeeping genes were the same in all. The autoradiographs were scanned with a MultiImager-Max system (Bio-Rad) and analyzed using Quantity One software (Bio-Rad) by constructing a grid with a window for each gene.

RT-PCR Analysis
RNA was isolated from human lung fibroblasts with TRIzol reagent (Invitrogen) following the manufacturer's instructions. RT-PCR was performed with the GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA) according to the kit's instructions. The following primers were used for PCR: T cell–specific surface glycoprotein (CD28), 5' forward primer 5'-tcatgtaccctccgccttac-3' and 3' reverse primer: 5'-gctggtaaggctttcgagtg-3'. Cytotoxic T-lymphocyte associated protein 4 (CD152), 5' forward primer 5'-caggtgacccaaccttcagt-3' and 3' reverse primer: 5'-cagtccttggatggtgaggt-3'. p-selectin glycoprotein ligand-1 (PSGL-1), 5' forward primer 5'-gcagagacctcaaaaccagc-3' and 3' reverse primer: 5'-tcagcagacattgcttcacc-3'. Leukemia inhibitory factor receptor (LIFr), 5' forward primer 5'-ttgctatcggaagcgagaat-3' and 3' reverse primer: 5'-acacgaccacatggttctca-3'. Macrophage colony-stimulating factor-1 receptor (MCSF-1r), 5' forward primer 5'-tgtgcaagaccatggtgaat-3' and 3' reverse primer: 5'-gtccacagcgttgagactga-3'. GAPDH, 5' forward primer 5'-acccagaagactgtggatgg-3' and 3' reverse primer: 5'-gggtcttactccttggaggc-3'. All amplifications shown here represent the product of twenty-eight cycles. Under the conditions used in these studies, plateau of control (GAPDH) and most amplicons was reached at 30 cycles.

Cytokine Treatment
Primary cultures of mouse adult lung fibroblasts were treated with either 1 ng/ml of TNF-{alpha}, 2.5 ng/ml of TGF-ß1, 1 ng/ml of IL-1ß, 1 ng/ml of IL-4 (all purchased from R&D Systems Inc, Minneapolis, MN), or a combination of two of each. Treated and untreated cells were exposed to the cytokines for 24–48 h. These experiments were conducted using either regular FBS or PDS. At the end of the culture period, the cells were immunostained for SRF as previously described in this manuscript.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Re-Expression of SRF{Delta}5 in Bleomycin-Induced Lung Fibrosis
SRF was easily detectable by immunoblot in lung tissues from control mice, and the level of expression did not change significantly with bleomycin treatment except on Day 17, when it was higher than in control lungs (Figure 1) . In contrast, SRF{Delta}5 was not detectable in control lungs, but was induced beginning on Day 7 after bleomycin treatment, with expression increasing to a maximum by Day 17. Levels of both isoforms gradually decreased thereafter, and by Day 21 there were no differences between control and bleomycin-treated lungs (not shown). It should be stressed that these studies were done using whole lung lysates. Therefore, changes in SRF/SRF{Delta}5, which only occur in a small number of cells (see below), are diminished by the presence of abundant normal lung cells.



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  		Figure 1. SRF isoforms in bleomycin-induced lung injury. Immunoblot on whole lung lysates showing expression of SRF{Delta}5 isoform first detected on Day 7 after bleomycin instillation and peaking on Day 17. SRF upregulation is noticeable only on Day 17 after treatment. Elevation of SM {alpha}-actin reflects the appearance and increment in myofibroblast numbers characteristic of the bleomycin model. Notice that the increase in both SRF/SRF{Delta}5 and SM {alpha}-actin are localized to relatively few cells in the lung and therefore are partially masked by the normal cells present in the lysates. Coomassie stain was performed to demonstrate equal loading.

 
SRF/SRF{Delta}5 Is Upregulated in Fibroblasts and Myofibroblasts
Immunohistochemical studies using anti-SRF/SRF{Delta}5 antibodies showed a generalized increase in SRF intensity in normal bronchial and vascular SM in Days 1 (not shown) and 3 after bleomycin treatment (Figure 2 , blood vessel wall in left panel). However, this was probably the result of bleomycin-mediated epitope unmasking, because no increase in SRF levels was detected in immunoblots (Figure 1). Few scattered clusters of peribronchial and perivascular fibroblasts with diffuse nuclear and cytoplasmic SRF positivity were identified in Day 3 lungs (Figure 2, right lower panel, arrow). On Day 7, and coinciding with SRF{Delta}5 appearance in Western blots, SRF/SRF{Delta}5 was immunodetected in clusters of fibroblasts within bleomycin-induced lesions (Figure 3 , upper panel). Interestingly, at this stage SRF/SRF{Delta}5 reactivity was stronger in the cytoplasm than in the nucleus and the cells exhibited enlarged "activated" nuclei. No myofibroblasts were found in these lesions, as indicated by absence of SM {alpha}-actin–positive cells (Figure 3, upper panel). SM {alpha}-actin–positive myofibroblasts were abundant on Day 14. At this stage SRF/SRF{Delta}5 immunoreactivity was found in the nuclei of myofibroblasts (Figure 3, middle panel). This pattern of SRF localization was still evident on Day 17 after lung bleomycin exposure (not shown). The SRF-positive cells immunoreacted with antibodies against TGF-ß1 and were negative for additional markers of SM differentiation, including desmin, SM myosin, and calponin (not shown). This immunohistochemical profile confirmed their myofibroblast nature. The lung parenchyma surrounding the lesion, as well as saline-treated control lungs, showed weak SRF immunoreactivity, which is not evident in this type of co-immunostaining studies (Figure 3, lower panel). Occasionally, however, blood vessels borderlining with or included in the lesion showed increment in SRF levels high enough to be detected as seen in Figure 3, upper panel.



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  		Figure 2. Lower panels: SRF-positive perivascular/peribronchial fibroblasts in early bleomycin-induced lung damage. Immunostaining demonstrated presence of fibroblasts with diffuse SRF positivity in perivascular area 3 d after bleomycin instillation (reddish color, arrow). In comparison, a matching control exposed to saline shows no cells at the perivascular site. Upper panels: low magnification fields showing the overall tissue architecture at this stage. No counterstain has been used in these studies. Magnification bar: 30 µm, lower panels; 300 µm, upper panels.

 


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  		Figure 3. SRF/SRF{Delta}5-positive fibroblasts and myofibroblasts in developing bleomycin-induced lesions. Co-immunostaining using FITC for SRF/SRF{Delta}5 (green fluorescence) and a nonfluorescent dye for SM {alpha}-actin (reddish brown) shows cytoplasmic enrichment for SRF/SRF{Delta}5 in clusters of enlarged, plump fibroblasts at lesion sites (upper panel). The inset represents a close-up of these cells to better show SRF/SRF{Delta}5 enrichment in their cytoplasm (n: nucleus). Notice positivity for both SM {alpha}-actin and nuclear SRF in SM cells forming blood vessel wall. Same immunohistochemical approach done after myofibroblast differentiation demonstrates nuclear localization of SRF/SRF{Delta}5 in lesional myofibroblasts and disappearance of cytoplasmic immunoreactive pattern (middle panel). Normal parenchyma (alveoli) is shown in the lower panel. Magnification bar: 30 µm.

 
Transient Transfection of SRF{Delta}5 and SRF into Lung Fibroblasts Induced Expression of Growth Factors and Cytokines Involved in Myofibroblast Differentiation
cDNA array analysis was done to begin to understand how SRF{Delta}5 and SRF may contribute to the pathogenesis of bleomycin-induced lung damage and emergence of the myofibroblast. Immunoblots showed successful transfection and, as previously shown (1, 2, 6), stimulation of SM {alpha}-actin by SRF (Figure 4) . SRF{Delta}5 did not alter SM {alpha}-actin expression in the 24-h-old primary cultures used for the cDNA analysis (Figure 4); however, a relatively mild SRF{Delta}5-induced increase in SM {alpha}-actin was observed after 3 d in culture (not shown). Notice that because these studies involved transient transfections, only a limited number of cells incorporated the plasmid construct within the total pool of cells, therefore the differences in gene expression were diminished by the presence of untransfected cells in the lysates. The average intensity of all the nine housekeeping genes on the array was used to normalize the results. The pattern of gene regulation was largely but not completely overlapping between SRF and SRF{Delta}5 (Table 1). As expected, SRF{Delta}5 and SRF upregulated mRNAs encoding for cell proliferation, survival, and migration (26), as well as SRF-related genes, such as SRF accessory protein 1A (SAP-1A) (not shown). SRF{Delta}5 and SRF upregulated many members of the TGF-ß superfamily of proteins, including TGF-ß1, consistent with myofibroblast transformation (Table 1). Interestingly, SRF and SRF{Delta}5 induced fibroblasts to express several proteins characteristically restricted to inflammatory cells (Table 1). De novo expression of some of these proteins was confirmed by RT-PCR (Figure 5) . No epithelial or endothelial cell markers were identified concurring with the RT-PCR studies done to verify absence of these two cell types in our primary cultures (not shown).



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  		Figure 4. Immunoblots showing transient transfection of SRF{Delta}5 and SRF plasmid constructs into mouse adult lung fibroblasts and their effect on SM {alpha}-actin levels. Twenty-four hours after transfection, SM {alpha}-actin is upregulated in cultures transfected with SRF, whereas SRF{Delta}5 has no effect on the former. Primary cultures of adult lung fibroblasts include a small number of SM cells, which account for the low levels of SM {alpha}-actin seen in all other lanes.

 

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  		TABLE 1 mRNAs upregulated over 2-fold in adult mouse lung fibroblasts by transient SRF{Delta}5 and SRF transfections

 


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  		Figure 5. RT-PCR confirming de novo expression of several inflammation-related proteins produced upon SRF{Delta}5 and SRF overexpression originally identified by array analysis.

 
Among the mRNAs upregulated by SRF not shown in this article were several ECM-degrading enzymes including messages for MMP-2, MMP-14, and urokinase-type plasminogen activator. Among the messages downregulated by SRF were those for tissue inhibitor of metalloproteinase-3 and type-1 plasminogen activator inhibitor (25). Because only few array results were confirmed by RT-PCR (Figure 5) (25), immunoblot (25), or immunohistochemistry (25) (TGF-ß1, not shown), it should be stressed that a single array experiment can give many false positive and false negative results.

Cytokine Effect on SRF Isoform Expression Pattern and Intracellular Distribution
Immunohistochemical studies demonstrated increased SRF/SRF{Delta}5 positivity in fibroblasts treated with TNF-{alpha} (Figure 6 , right upper panel) and IL-1ß (not shown). In those cases most of the immunoreactivity was localized to the cytoplasm of treated cells. In cells exposed to TGF-ß1, SRF localized mainly to the nucleus but remained rather weak in intensity (Figure 6, left lower panel). IL-4 did not affect SRF/SRF{Delta}5 immunoreactivity (not shown). The combination of TNF-{alpha} and TGF-ß1 resulted in both increased levels of SRF/SRF{Delta}5 and cytoplasmic/nuclear translocation (Figure 6, right lower panel). This double effect was not seen with other cytokine combinations (not shown).



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  		Figure 6. Intracellular SRF/SRF{Delta}5 is modulated by cytokines involved in bleomycin-induced lung injury. Immunohistochemical studies showing increased SRF/SRF{Delta}5 positivity in fibroblasts treated with TNF-{alpha} (upper right), compared with controls (upper left). Notice that most of the immunoreactivity is localized in the cytoplasm. In cells treated with TGF-ß1, SRF is mainly localized in the nucleus (lower left); however, the immunostaining remains weak, as in control cells. The combination of TNF-{alpha} and TGF-ß1 results in increased levels of SRF/SRF{Delta}5 and cytoplasmic/nuclear translocation (lower right). A control in which the primary antibody (anti-SRF/SRF{Delta}5) was omitted is shown in the two small right panels, the upper one representing light microscopy, as the rest of the figure, and the lower one representing Normanski differential interference contrast microscopy (DIC). Magnification bar: 60 µm.

 
Immunoblots demonstrated that TNF-{alpha} (Figure 7) and IL-1ß (not shown) induced production of SRF{Delta}5, but did not stimulate SM {alpha}-actin synthesis. TGF-ß1 treatment did not alter SRF levels or induced expression of SRF{Delta}5 in Western blots, but as expected (27, 28), it upregulated SM {alpha}-actin synthesis (Figure 7). IL-4, another cytokine known to induce myofibroblast differentiation (19), did not affect SRF expression (not shown). The combination of TNF-{alpha} with TGF-ß1 induced SRF{Delta}5, upregulated SRF, and produced maximal stimulation of SM {alpha}-actin expression (Figure 7).



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  		Figure 7. SRF and SRF{Delta}5 levels are regulated by cytokines involved in bleomycin-induced lung injury. Immunoblots showing induction of SRF{Delta}5 by TNF-{alpha} but not by TGF-ß1. The latter does not alter SRF levels or induce expression of SRF{Delta}5, but as previously shown in the literature, it upregulates SM {alpha}-actin synthesis. The combination of TNF-{alpha} with TGF-ß1 induces SRF{Delta}5, upregulates SRF, and produces maximal SM {alpha}-actin stimulation. Primary cultures of adult lung fibroblasts include a small number of SM cells, which account for the low levels of SM {alpha}-actin seen in all other lanes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we have shown that SRF and mainly its truncated isoform SRF{Delta}5 are highly expressed in fibroblasts and myofibroblasts found in lung lesions of bleomycin-treated mice. The earliest change observed was the presence of SRF/(SRF{Delta}5?)-positive fibroblasts in perivascular/peribronchial areas, detected 3 d after bleomycin instillation. Although the antibody does not discern between SRF{Delta}5 and SRF, for it binds to both, this finding is of potential significance because bleomycin-induced fibrotic lesions originate at these sites (11, 29). On Day 7 after treatment and coinciding with the appearance of SRF{Delta}5 in immunoblots, SRF/SRF{Delta}5 was found enriched in the cytoplasm of scattered enlarged "activated" fibroblasts within developing lesions. This changed on Day 14, when myofibroblasts were identified. At this time SRF and SRF{Delta}5 levels were significantly upregulated and were immunodetected mainly within myofibroblast nuclei. All these observations suggested a role for SRF{Delta}5 and SRF in the development of bleomycin-induced lung lesions.

cDNA array analysis of lung fibroblasts transiently transfected with SRF{Delta}5 and SRF was then performed to better understand their potential pathogenic role. As expected (26), SRF and SRF{Delta}5 upregulated the expression of mRNAs coding for cell proliferation and survival, as well as SRF-related genes. Several members of the TGF-ß superfamily of proteins (30) were upregulated by SRF{Delta}5 and SRF. However, there were differences in the induction patterns elicited by each isoform. For example, SRF{Delta}5 had a lesser effect on TGF-ß1, the key growth factor involved in myofibroblast differentiation (28), whereas it induced comparatively more activin ß subunit, which plays no role in SM or myofibroblast differentiation (31). SRF, on the other hand, had a stronger effect on TGF-ß1 and also induced follistatin, which stimulates muscle growth (32).

SRF and SRF{Delta}5 induced fibroblasts to produce several chemokines involved in inflammation. These included monocyte chemoattractant protein 3, which attracts and activates a great variety of inflammatory cell types (33); macrophage colony-stimulating factor, which stimulates alveolar macrophage proliferation (34); and IL-11, a cytokine involved in lung inflammation and fibrosis (35).

Furthermore, SRF{Delta}5 and SRF induced fibroblasts to express several intracellular/cell membrane proteins characteristically restricted to inflammatory cells, such as T cell–specific surface glycoprotein (CD28) (36), cytotoxic T-lymphocyte–associated protein 4 (CD152) (37), and PSGL-1 (38), usually expressed by neutrophils, monocytes, and dendritic cells (38). The novo expression of these proteins was confirmed by RT-PCR. To the best of our knowledge, none of them have been reported in other cell types, including fibroblasts and myofibroblasts.

There were few mRNAs that were upregulated or induced by SRF{Delta}5 alone. Among them was CD44, which is a cell-surface adhesion molecule and hyaluronan receptor (39) originally identified in T cells (40) but later found in noninflammatory cell types (41, 42). CD44 is involved in recruiting T cells to inflammatory sites (40, 43). Moreover, it has been recently shown that mice null for cd44 have impaired activation of TGF-ß1 (44). An additional protein induced only by SRF{Delta}5 was the cytoskeletal protein MOESIN-ezrin-radixin-like protein, also known as schawnnomin or neurofibromatosis 2. This protein links actin microfilaments to the plasma membrane and transduces growth signals via the cytoskeleton (45). Interestingly, PSGL1, which is induced by SRF and SRF{Delta}5, co-localizes with ezrin/redixin/moesin at the trailing edge of neutrophils and is involved in the rolling of these cells on activated endothelium (38). Finally, SRF{Delta}5 stimulated Crk, which enhances cell motility by participating in actin membrane ruffles (46). Altogether, these findings seemed to indicate that SRF{Delta}5 is mainly involved in cell migration to developing lesion sites, and in the initiation of production and activation of TGF-ß1, both steps are essential for triggering myofibroblast differentiation, whereas SRF mostly stimulates cell proliferation and survival and the development of a mature myofibroblast phenotype.

Although reported elsewhere, it is important to remark that only a few genes were downregulated by SRF and SRF{Delta}5, the most relevant being tissue inhibitor of metalloproteinase-3 and type-1 plasminogen activator inhibitor (25), which were affected only by SRF. SRF also stimulated expression of several MMPs and other ECM-degrading enzymes (25), suggesting that a major difference between SRF and SRF{Delta}5 is that SRF creates a pro-proteolytic imbalance that favors ECM degradation (25).

The main cytokines involved in early bleomycin-induced tissue damage are TNF-{alpha} and IL-1ß (10), whereas the critical cytokines involved in the development of myofibroblasts and fibrosis are TGF-ß1 (27, 28) and, perhaps, IL-4 (19, 47). In functional studies using mouse adult lung fibroblasts, we found that the combination of TGF-ß1 and TNF-{alpha} caused all the SRF changes observed in vivo and resulted in maximal SM {alpha}-actin upregulation.

In summary, our studies suggested that SRF and SRF{Delta}5 play a role in bleomycin-induced lung fibrosis by stimulating myofibroblast differentiation and their expression of a broad range of proteins involved in cell migration, proliferation, and inflammation. Furthermore, we found that SRF and SRF{Delta}5 levels and intracellular localization are regulated, at least in part, by cytokines found at the lesion site.


   Acknowledgments
 
This work was supported by NHLBI grants HL-48730 and HL-67100, a grant from the Children's Research Center of Michigan (to L.S.) and NIH grants HL28737 and HL52285 (to S.H.P.).


   Footnotes
 
* Both authors contributed equally to the results of this work. Y.Y. is currently at Human Genome Sciences, Inc., Rockville, Maryland. Back

Received in original form December 23, 2002

Received in final form April 22, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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