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Transforming Growth Factor- Receptor Type 1 (TGFRI) Kinase Activity but Not p38 Activation Is Required for TGFRI-Induced Myofibroblast Differentiation and Profibrotic Gene Expression

Scios Inc., Fremont, California (A.M.K., N.J.G., Y.W., D.D., Y.-W.L., G.O., D.Q., A.L., K.M., T.-T.T., J.Y.M., A.M., S.D., S.C., A.A.P., L.S.H.); and Nebraska Medical Center, Omaha, Nebraska (F.-Q.W., X.L., S.I.R.)

Received December 9, 2005; accepted April 4, 2006

	Abstract

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Transforming growth factor- (TGF) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGF also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGF-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGF-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.

TGF, a major player in the conversion of fibroblasts to myofibroblasts, is a pleiotropic growth factor involved in multiple biological processes including cell proliferation, fibrosis, tissue repair, inflammation, apoptosis, cell differentiation, cell adhesion, and motility (Sporn and Roberts, 1992; Massague et al., 1994; Grande, 1997). TGF levels are elevated in the bronchoalveolar lavage fluid of patients with pulmonary fibrosis (Kuroki et al., 1995; Ludwicka et al., 1995) and in the lungs of animals with experimentally induced lung fibrosis (Coker et al., 1997). TGF signals through heteromeric receptor complexes consisting of type I (TGFRI) and type II (TGFRII) serine-threonine kinase receptors. Upon ligand binding, TGFRII transphosphorylates TGFRI. The activated receptor then phosphorylates and activates members of the SMAD family of proteins. Receptor SMADs subsequently bind to coSMAD4 and translocate into the nucleus, where they regulate the transcriptional response of target genes.

Besides signaling through SMADs, several additional pathways have been shown to mediate the downstream signaling from TGFRI. These include the p38 mitogen-activated kinase (MAPK) (Bhowmick et al., 2001; Bakin et al., 2002), the c-Jun NH₂-terminal kinase (Atfi et al., 1997), the extracellular-regulated kinase (Frey and Mulder, 1997), and the phosphatidylinositol kinase pathways (Bakin et al., 2000). The p38 pathway is involved in a variety of biological responses to the TGF signaling, such as migration of smooth muscle cells (Hedges et al., 1999), neuronal differentiation of PC-12 cells (Iwasaki et al., 1999), chondrogenesis of ATDC-5 cells (Nakamura et al., 1999), and cardiomyocyte differentiation (Monzen et al., 1999). Despite the numerous implications of p38 signaling in the TGF pathway, the significance of TGF-mediated p38 signaling in pulmonary fibrosis and myofibroblast transformation is not well defined.

In this study, we used a combination of cDNA arrays and small molecule inhibitors of TGF-R1 and p38 MAP kinase to begin to delineate the molecular events downstream of TGF-R1. We identify a number of TGF-regulated genes and assess which genes are regulated by p38. In addition, we show that small molecule inhibitors of TGFRI, but not p38, block SMAD signaling, inhibit an in vitro model of fibrotic tissue remodeling (fibroblast mediated collagen gel contraction), and prevent the transformation of fibroblasts to myofibroblasts in vivo and in vitro, as measured by expression of -smooth muscle actin.

	Materials and Methods

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Cell Culture and Inhibitor Reagents. Three lots of primary human lung fibroblast (HLF) cells, derived from a 40-year-old woman (donor A, lot 8F162), a 10-year-old boy (donor B, lot 17423), and a 51-year-old woman (donor C, lot 4F0512), were provided by Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Realtime RT-PCR experiments were performed on samples from all donors. The microarray and gel contraction experiments were performed on cells from donor A, and the ELISA and Western blot data were assayed using samples from donors A and C. Cells were seeded at 4 x 10⁵ cells (passage 4) in 100-mm² dishes and cultured in complete fibroblast growth medium (FGM; Cambrex Bio Science Walkersville, Inc.). The next day, medium was changed to FGM without serum or fibroblast growth factor but supplemented with 0.2% bovine serum albumin and 50 µg/ml vitamin C. Cells for realtime RT-PCR and microarray were serum-deprived for 24 h before treating with 5 ng/ml TGF1 (R&D Systems, Minneapolis, MN) in the presence or absence of 400 nM SD-208 or 400 nM SD-282. Concentrations of inhibitors were chosen based on IC_50s in HLF cell-based assays (data not shown and Fig. 2; IC₅₀, 70-80 nM). Induction with TGF was carried out for various times (7.5, 24, and 72 h) after which RNA was harvested by lysing the cells in RLT buffer (QIAGEN, Valencia, CA) and frozen at -80°C.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Effects of SD-208 and SD-282 on TGF-induced SMAD2/3 phosphorylation in HLF cells. Cells were treated with TGF for 30 min in the presence of multiple concentrations (0-400 nM) of SD-208 (A and C) or SD-282 (B and D). SMAD phosphorylation was measured by ELISA (A and B) and Western blot analysis (C and D) using antibodies for SMAD2/3 and SMAD2, respectively. Analysis are presented as mean ± S.D. from three or four separate experiments. Relative responses for each curve (C and D) were determined by defining the p-Smad response for TGF-treated + 0 nM compound as 100%. Band densities (C and D) were quantitated with the use of Image Quant 5.2. Statistical significance (***, P < 0.001; **, P < 0.01; and *, P < 0.05) was determined for each treatment relative to TGF-treated + 0 nM (below the line) and relative to the next lower inhibitor concentration (above the line).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Structures of SD-208 and SD-282.

Western Blot and ELISA Analysis. HLF cells were serumstarved for 24 h in FGM (Cambrex Bio Science Walkersville, Inc.) containing no serum or FGF but supplemented with 0.2% BSA and 50 µg/ml vitamin C. To analyze SMAD phosphorylation, cells were treated with 2 ng/ml TGF1 for 30 min in the presence of increasing concentrations of SD-208 or SD-282 inhibitor as indicated. To analyze induction of -smooth muscle actin, CTGF, and PAI-1, cells were treated with 2 ng/ml TGF1 for 7, 24, or 72 h in the presence of 400 nM SD-208 or SD-282. Total cell lysates were prepared in MPER buffer (mammalian protein extraction reagent; Pierce, Rockford, IL) or in radioimmunoprecipitation assay (10 nM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS; Roche, Indianapolis, IN) containing protease and phosphatase inhibitor cocktails. Aliquots of the cell lysates were fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). The membranes were blocked against nonspecific binding using 4% skim milk. Proteins were detected using specific primary antibodies and peroxidase-conjugated secondary antibodies. The antibody against phospho-SMAD2 was from Cell Signaling Technology (Beverly, MA); the mouse monoclonal antibody against SMAD2 was purchased from BD Biosciences (Palo Alto, CA); the CTGF polyclonal antibody was custom made (U. Schellenberger, Scios Inc.); the monoclonal antibody against -smooth muscle actin was purchased from Sigma (St. Louis, MO); the mouse monoclonal antibody against GAPDH was from Biogenesis (Poole, Dorset, UK); and the mouse monoclonal antibody against vimentin was from Affinity Bioreagents (Golden, CO). The blots were visualized by the SuperSignal West Femto detection system (Pierce) or the Amersham ECL detection system followed by quantitation using Image Quant 5.2 (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Phospho-SMAD ELISA assays were performed in 96-well platescoated with anti-SMAD2/3 monoclonal antibodies (100 ng/well; BD Biosciences) for 18 h at 4°C or2hat room temperature. To block nonspecific binding, excess antibody was removed and the wells were treated with blocking buffer (0.3% BSA/PBS) for 2 h at room temperature. The wells were rinsed three times with wash buffer (0.5% Tween 20/PBS), and cell lysates (10 µg of total protein) were added to each well and incubated overnight at 4°C. Wells were rinsed three times with wash buffer before adding a polyclonal anti-phospho-SMAD2/3 antisera diluted in 2% BSA/0.5% Tween 20/PBS. After a 2-h incubation at room temperature, wells were washed three times with wash buffer before developing with tetramethyl benzidine (Sigma). Reactions were stopped with 0.5 N H₂SO₄ after a 5- to 30-min incubation, and plates were read at an optical density of 450 nm in a SpectraMax 250 plate reader (Molecular Devices, Sunnyvale, CA). For PAI-1 ELISA analysis, Imulyse (Biopool International, Ventura, CA) kits were used according to the manufacturer's instructions.

In Vitro Immunofluorescence Analysis. To examine the TGF-induced nuclear translocation of SMAD2, HLF cells were grown to 50 to 80% confluence in Lab-Tek Chamber Slides (Nalge Nunc International, Naperville, IL). Cells were serum-starved and treated with TGF1 (2 ng/ml) and inhibitors as described above. After treatment, cells were washed with PBS and fixed for 15 min with 4% paraformaldehyde in PBS. Cells were then treated with 0.1% saponin in PBS for 10 min. After washing off the detergent, fixed cells were incubated with primary antibodies overnight at 4°C. Specific antibodies for SMAD2 were from Zymed Laboratories (South San Francisco, CA). After extensive washing, the biotinylated antirabbit IgG and fluorescein avidin D secondary antibodies (Vector Laboratories, Burlingame, CA) were added. The fluorescence images of SMAD2 were visualized by a Nikon microscope using Image-Pro plus 4.5 software (Media Cybernetics, Inc., Silver Spring, MD).

Collagen Gel Contraction. Native type I collagen (rat tail tendon collagen) was extracted from rat tail tendons by a method published previously (Bell et al., 1979; Mio et al., 1996). In brief, tendons were excised from rat tails without tendon sheath and other connective tissues. Repeated washing with Tris-buffered saline was followed by dehydration and sterilization with 50%, 75%, 95% and pure ethanol. Type I collagen was then extracted in 6 mM hydrochloric acid at 4°C. The supernatant was harvested by centrifugation at 3000g for 1 h at 4°C. Collagen concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution.

Gels were prepared using a method described previously (Mio et al., 1996, 1998) that involves mixing rat tail tendon collagen, distilled water, and 4x Dulbecco's modified Eagle's medium so that the final mixture resulted in a physiologic ionic strength, 1x Dulbecco's modified Eagle's medium, and a pH of 7.40. Cells were trypsinized (trypsin-EDTA; 0.05% trypsin, 0.53 mM EDTA-4Na; Invitrogen) and suspended in 10 ml of complete FGM and counted with a Coulter Counter (Beckman Coulter, Fullerton, CA). HLF cells were pelleted and resuspended in basal FGM without serum or other growth factors at a density of 10⁷ cells/ml. Cells were then mixed with the neutralized collagen solution so that the final cell density in the collagen solution was 5 x 10⁵ cells/ml, and the final concentration of collagen was 0.75 mg/ml. Aliquots (0.5 ml/well) of the mixture of cells in collagen were cast into each well of 24-well tissue culture plates (BD Biosciences). After gelation was completed, within 20 min at room temperature, the gels were gently released from the 24-well tissue culture plates and transferred into 60-mm tissue culture dishes (three gels in each dish) that contained 5 ml of freshly prepared basal FGM with or without TGF-1 (200 pmol/l) or inhibitors. The gels were then incubated at 37°C in a 5% CO₂ atmosphere for 2 days, and the area of each gel was measured with an Optomax V image analyzer (Optomax, Burlington, MA) daily. Data were expressed as the percentage area compared with the original gel size.

Histology and Immunohistochemistry in the Rat Bleomycin Model. Sprague-Dawley rats were administered bleomycin for 14 days. Rats were intubated and aerosolized with 200 µl of saline or 1.0 unit of bleomycin per rat (three to four rats per group). One day after bleomycin challenge rats were treated twice daily with vehicle or vehicle plus SD-208 (60 mg/kg in 1% methyl cellulose) or SD-282 (60 mg/kg in 1% PEG-400). Inhibitor doses were chosen based on efficacious doses from our previous studies (Li et al., 2004; Bonniaud et al., 2005). In addition, the inhibitor doses used in this study have been shown to have pharmacological effects in the lungs of bleomycin-challenged rats as demonstrated by the inhibition of CTGF mRNA by SD-208 and of COX2 mRNA by SD-282 (data not shown). Lungs were removed en bloc; they were inflated with 4% formalin at a constant pressure of 15 cm of water and then fixed in 10% formalin for 48 h. The left lung was cut perpendicular to the tracheobronchial tree into sections. Tissue sections were processed, embedded in paraffin, cut into 5-µm sections, and immunohistochemically stained using mouse antibodies against -smooth muscle actin (Chemicon International, Inc., Temecula, CA). Negative control sections were run in parallel with normal mouse IgG2 diluted to the same concentration as the primary antibody (data not shown). The number of myofibroblasts was measured under the Nikon E600 light microscope equipped with a Spot digital camera at magnification of 400x for 36 to 40 fields (for each animal). Positively stained smooth muscle cells surrounding major airways and blood vessels were excluded from the analysis.

Measurement of Hydroxyproline Content. To quantify lung collagen content as an indicator of pulmonary fibrosis, the hydroxyproline content in whole lungs was measured in all animals (seven to eight animals per group) according to published methods (Woessner, 1961). In brief, lung samples were minced into fine pieces with a scissors and homogenized thoroughly in 15 ml of 1x PBS with a Polytron homogenizer (Kinematica, Basel, Switzerland). One milliliter of whole homogenate was precipitated with 0.25 ml of ice-cold 50% (w/v) trichloroacetic acid. The precipitate was hydrolyzed in 2.0 ml of 6 N HCL for 18 h at 110°C. Hydroxyproline content was determined after neutralization. The results were calculated as micrograms of hydroxyproline per whole lung using hydroxyproline standards from Sigma (St. Louis, MO).

cDNA Microarray. Gene expression profiles were determined from cDNA microarrays as described previously (Kapoun et al., 2004). In brief, arrays containing 8600 elements were derived from clones isolated from normalized cDNA libraries or purchased from ResGen (Invitrogen). Clones were sequence verified, and differentially expressed genes were reconfirmed by NCBI BLAST analyses. Differential expression values were expressed as the ratio of the median of background-subtracted fluorescent intensity of the experimental RNA to the median of background-subtracted fluorescent intensity of the control RNA. For ratios greater than or equal to 1.0, the ratio was expressed as a positive value. For ratios less than 1.0, the ratio was expressed as the negative reciprocal (e.g., a ratio of 0.5 =-2.0). Differential expression ratios were determined as the mean of the two values from dye-swapped duplicates. Expression data were rejected if neither channel produced a signal of at least 2.0-fold over background. Statistically significant differential expression threshold values were determined according to the method of Yang et al. (2002). Hierarchical clustering was used to visualize the data and to group genes into similar expression patterns (Spotfire, Somerville, MA). The data were prepared for clustering by using the log base 10 of the median expression values and then normalized by the Z-score method within Spotfire.

mRNA Isolation, Labeling, and Hybridizations. Total RNA was extracted from cells using QIAGEN's RNeasy kit (Valencia, CA). RNA was amplified using a modified Eberwine protocol (Kapoun et al., 2004) that incorporated a poly(A) tail into the amplified RNA. Fluorescently labeled cDNA probes were generated by reverse transcription of 4 µg of RNA with SuperScript II (Invitrogen) using anchored dT primers in the presence of Cy3 or Cy5 dUTP as described previously (Kapoun et al., 2004). Hybridization of each fluorophore was quantified using an Axon GenePix 4000A scanner.

Real-Time RT-PCR. Real-time RT-PCR was performed in a twostep manner. cDNA synthesis and real-time detection were carried out in a PTC-100 Thermal Cycler (MJ Research Inc., Waltham, MA) and an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), respectively. Random hexamers (QIAGEN) were used to generate cDNA from 200 ng of RNA as described in Applied Biosystems User Bulletin 2. TaqMan PCR Core Reagent Kit or TaqMan Universal PCR Master Mix (Applied Biosystems) were used in subsequent PCR reactions according to the manufacturer's protocols. Relative quantitation of gene expression was performed using the relative standard curve method.

Sequence specific primers and probes were designed using Primer Express software (ver. 2; Applied Biosystems). Sequences of primers and probes can be found in Supplemental Table E1. Expression levels were normalized to 18 S rRNA. All real-time RT-PCR reactions were performed in triplicate on each of the three biological replicates from each donor.

Statistical Analysis. The experiments were usually performed 3 to 4 times with similar results. Significance was tested by one- or two-tailed analysis of variance followed by Bonferroni's correction using PRISM4 software (GraphPad Software Inc. San Diego, CA) unless otherwise indicated.

	Results

Top Abstract Materials and Methods Results Discussion References

 
The TGFRI Kinase Inhibitor SD-208 Blocks SMAD Signaling in HLF Cells
The initial characterization of SD-208 and SD-282 in cellfree assays led to their identification as a potent inhibitors of TGFRI and p38 MAPK. SD-208 has an IC₅₀ of 49 nM based on direct enzymatic assay of TGFRI kinase activity with specificity of at least >17-fold over members of a panel of related protein kinases (Table 1). SD-282 is a small molecule, ATP-competitive, specific inhibitor of p38 and p38 MAP kinase, and demonstrates 14-fold selectivity for p38 versus p38 MAP kinase (IC₅₀, 1.6 and 23 nM, respectively; Table 2).

View this table: [in this window] [in a new window]   TABLE 1 Specificity of SD-208 for TGFR1 kinase activity Data are expressed as IC50 or as percentage inhibition at 50 µM SD-208 based on direct enzymatic assay.

As SMAD phosphorylation and nuclear translocation are well characterized steps in TGF signaling, we tested the ability of pharmacological inhibitors of TGFRI kinase and p38 MAPK to modulate these events. HLF cells were treated with TGF for 30 min in the presence of either a small molecule inhibitor of TGFRI (SD-208) or of p38 MAP kinase (SD-282) for 30 min.

ELISA and Western blot analysis showed that SD-208, but not SD-282, inhibited TGF-induced SMAD2/3 and SMAD2 phosphorylation, respectively, in a dose-dependent manner (0-400 nM; Fig. 2). In addition, SD-208 blocked TGF-induced nuclear translocation of SMAD2 in HLF cells; however, SD-282 had no effect (Fig. 3). These results show that SD-208 is an effective inhibitor of TGF-induced SMAD signaling and suggest that p38 has no direct role in SMAD-mediated signaling during myofibroblast differentiation.

View larger version (126K): [in this window] [in a new window]   Fig. 3. Effects of SD-208 and SD-282 on TGF-induced SMAD2 nuclear translocation in human lung fibroblasts. Human lung fibroblast cells were treated in the presence or absence of TGF for 30 min with or without SD-208 (200 nM) or SD-282 (200 nM).

The TGFRI Kinase Inhibitor SD-208 Inhibits Myofibroblast Differentiation and Collagen Gel Contraction in HLF Cells

View larger version (31K): [in this window] [in a new window]   Fig. 4. Inhibition of -smooth muscle actin protein expression. Protein levels of -smooth muscle actin were measured by Western blot analysis from HLF cells treated for 24 h (A and B) and 72 h (C and D) with TGF in the presence of either SD-208 (400 nM) or SD-282 (400 nM). The histograms in A and C represent mean data points from two separate experiments, each including triplicate biological replicates (n = 5-6, mean ± S.D.). The data are normalized to GAPDH and expressed as a percentage compared with the TGF-treated samples. B and D are representative Western blots showing -smooth muscle actin (-SMA) and GAPDH protein staining. ***, P < 0.001; **, P < 0.01; *, P < 0.05 versus TGF.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effects of SD-208 and SD-282 on TGF-induced collagen gel contraction. HLF cells were cast in three-dimensional collagen gels. Gels were released into media with and without TGF in the presence or absence of SD-208 (A) or SD-282 (B). After 2 days of culture, the area of the floating gels was measured with an image analyzer. Data are presented as a means ± S.E. of three replicate gels for each condition; ***, P =<0.001 versus TGF-treated control (SF-FBS). Statistical significance (P =<0.001) between inhibitor doses was observed for the following comparisons: the TGF + SD-208 group (25 versus 100 nM, 25 versus 200 nM, 50 versus 100 nM, 50 versus 200 nM); the SD-208 group (25 versus 200 nM), and the TGF+SD-282 group (50 versus 100 nM).

TGFRI Kinase Inhibitor SD-208 Inhibits Myofibroblast Differentiation and Collagen Deposition in Vivo in Bleomycin-Treated Rat Lungs

Saline-treated rats stained positive for -smooth muscle actin around major airways and blood vessels, whereas bleomycin-treated animals had significantly higher numbers of myofibroblasts (-smooth muscle actin-positive cells) at the fibroblastic foci (Fig. 6). SD-208, but not SD-282, significantly reduced the number of cells staining positive for -smooth muscle actin (Fig. 6). In a repeat experiment, lung hydroxyproline content was measured in SD-208-treated animals compared with vehicle control (Fig. 7). Hydroxyproline content is a well characterized assessment of the amount of fibrosis in tissues (Woessner, 1961). Bleomycin-treated rats that were dosed with 60 mg/kg SD-208 showed a significant decrease in the total hydroxyproline per lung compared with the 1% methyl cellulose control (^***, p < 0.001). These results demonstrate that TGF-receptor blockade, but not p38 inhibition, attenuates myofibroblast conversion and collagen deposition in an in vivo model of pulmonary fibrosis.

View larger version (83K): [in this window] [in a new window]   Fig. 6. Histology of lungs from bleomycin-treated rats ± SD-208 and SD-282. The animals were sacrificed 14 days after instillation of bleomycin or vehicle. The lungs were stained by immunohistochemistry with -smooth muscle actin. A, saline + 1% MC; B, Bleo + 1% MC; C, Bleo + SD-208 (60 mg/kg); D, saline + 1% PEG-400; E, Bleo + 1% PEG-400; F, Bleo + SD-282 (60 mg/kg). Magnifications shown for A-F are 400x. Histograms (bottom) represent the number of myofibroblasts at the fibroblastic foci (mean ± S.D. for 36-40 fields from each of three to four animals). Statistical significance (P < 0.01, unpaired student's t test) was achieved for the SD-208 versus vehicle group.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Collagen deposition as a measure of fibrosis in bleomycin-treated rats. The animals were sacrificed 14 days after instillation of bleomycin or vehicle. Hydroxyproline content in whole lungs was measured in all animals (seven to eight animals per group). Bleomycin-treated rats dosed with 60 mg/kg SD-208 showed a significant decrease in the total hydroxyproline per lung compared with the 1% methyl cellulose control (***, P < 0.001).

TGFRI Kinase Blockade Affects a Majority of TGF-Responsive Genes in HLF Cells
We employed SD-208 and SD-282 together with cDNA microarray analysis to dissect the molecular signaling events downstream of the TGF pathway. Using these compounds, we tested the ability of each compound to affect TGF-regulated gene expression profiles in treated HLF cells at various times. A time course was performed to comprehensively investigate the range of TGF response genes, including the acute early gene changes as well as the later TGF events. Fluorescently labeled cDNAs were generated from four experimental groups at multiple time points (1, 7.5, 24, and 72 h): unstimulated cells (control), TGF-treated cells, TGF- and SD-208-treated cells, and TGF- and SD-282-treated cells. The following hybridizations were performed in duplicate: control versus TGF, TGF versus TGF and SD-208, and TGF versus TGF and SD-282. The 1-h samples were omitted from the inhibitor analysis, because TGF affected a small number of genes. As a result, this time point would not contribute significantly to a functional analysis of inhibitor-affected genes.

A time course of TGF-responsive genes was identified (Fig. 8A). In terms of the number of genes affected by TGF, maximum changes were observed at 24 h (640 up- and 898 down-regulated). SD-208 had significant effects on gene expression induced by TGF; at 7.5, 24, and 72 h, the percentage of effected genes was 77, 84, and 58%, respectively (Fig. 8B). In contrast, at these time points, SD-282 inhibited between 10 and 18% of TGF-regulated genes (Fig. 8B). Most genes inhibited by SD-282 are also affected by SD-208 (Fig. 8C), consistent with the expectation that the blockade of the TGFRI signaling affects the p38 MAPK pathway in TGF-simulated cells.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Microarray analysis in TGF-treated HLF cells. A, gene expression changes induced by TGF and in HLF cells at 1, 7.5, 24 and 72 h. Histograms show the number of gene expression changes that were up-regulated and down-regulated by TGF treatment. Hybridizations using fluorescently labeled cDNA probes compare untreated (control) to TGF-treated cells. Histogram bars: up-regulated genes (black) and down-regulated genes (gray). B, effects of SD-208 (400 nM) and SD-282 (400 nM) on TGF-induced gene expression. Hybridizations using fluorescently labeled cDNA probes compare TGF-treated to TGF + inhibitor-treated cells at 7.5, 24, and 72 h. See Materials and Methods for details related to statistical significance. Histogram bars: no inhibitor effect (white) and inhibitor effect (black). C, Venn diagram showing overlap of TGF-induced gene expression changes affected by SD-208 and/or SD-282 at 7.5, 24, and 72 h.

Gene Expression Clustering
Genes were grouped according to functional categories by using a combination of gene expression clustering and functional annotations (Fig. 9).

View larger version (56K): [in this window] [in a new window]   Fig. 9. Gene expression clusters in HLF cells: fibrosis and ECM (A), inflammation (B), cytoskeletal (C), cell proliferation (D), apoptosis and cell survival (E), and TGF and p38 MAPK pathway (F). Each column represents the results from duplicate hybridizations: 1) control versus TGF, 1 h; 2) control versus TGF, 7.5 h; 3) control versus TGF, 24 h; 4) control versus TGF, 72 h; 5)TGF versus TGF + SD-208, 7.5 h; 6) TGF versus TGF + SD-208, 24 h; 7) TGF versus TGF + SD-208, 72 h; 8) TGF versus TGF + SD-282, 7.5 h; 9) TGF versus TGF + SD-282, 24 h; and 10) TGF versus TGF + SD-282, 72 h. Normalized data values depicted in shades of red and green represent elevated and repressed expression, respectively. Data were generated using the hierarchical clustering algorithm contained in Spotfire software. See Supplemental Table E2 for gene identities and expression values.

Fibrosis and Extracellular Matrix Genes. A cluster of genes involved in fibrosis and extracellular matrix (ECM) deposition were regulated in response to TGF (Fig. 9A). A majority of these genes were up-regulated by TGF. Representative examples include CTGF, fibronectin, PAI-1, TIMP3, THBS2, and several members of the Collagen gene family. All of these TGF-stimulated gene events were reversed by SD-208. SD-282 also showed a modest reversal of some of the genes in this cluster, such as COMP (cartilage oligomeric matrix protein), TIMP3, and IL11. These results show that SD-208 globally opposed TGF induced fibrotic gene expression in human lung fibroblasts, whereas SD-282 affected only a subset of the fibrogenic genes.

Inflammation Genes. Genes involved in inflammatory processes were elevated and repressed by TGF in HLF cells (Fig. 9B). TGF stimulated pro-inflammatory genes including COX2, COX1, IL6, interleukin 1 receptor accessory protein (IL1RAP), bradykinin receptor B2 (BDKRB2), tumor necrosis factor, -induced protein 6 (TNFAIP6), and TNF superfamily member 4 (TNFSF4). At the same time, TGF also downregulated pro-inflammatory genes such as CCL13 (MCP-4), CXCL7 (MCP-3), CCL8 (MCP-2), and CXCL1 (GRO1). The effects of TGF on these genes increased over time; repression by TGF was not seen until 24 h (4-6-fold) whereas by 72 h, the down-regulation increased to 8- to 11-fold. The TGF effects on all these gene regulatory events were opposed by SD-208. It is noteworthy that 30% of the TGF-regulated inflammatory genes were also reversed by SD-282. The genes, which are reversed by both inhibitors, include COX2, IL1RAP, TNFAIP6, and TNFSF4. Together, these data suggest that SD-208 and SD-282 attenuate TGF-induced pro-inflammatory gene responses in human lung fibroblasts.

Cytoskeletal Genes. A group of cytoskeletal genes was found to be regulated by TGF (Fig. 9C). A majority of these genes are up-regulated by TGF. Representative genes from this cluster include known markers of myofibroblasts such as -smooth muscle actin 2 (ACTA2), -skeletal muscle actin 1 (ACTA1), and nonmuscle myosin heavy chain 9 (MYH9). Many of the genes in this group are novel TGF-responsive genes. For example, TGF up-regulated dystrophin (DMD). DMD is involved in muscle contraction, which is an additional property that myofibroblasts acquire. SD-208, the TGFRI inhibitor, but not SD-282, the p38 MAPK inhibitor, opposed a majority of the TGF-induced cytoskeletal gene changes in this cluster. This result is consistent with the -smooth muscle actin Western data showing that SD-208 blocks the transformation of fibroblasts to myofibroblasts. Furthermore, the temporal induction patterns of genes contributing to myofibroblast differentiation and contraction were up-regulated between 7.5 and 24 h, consistent with our observation by Western analysis that -smooth muscle actin expression becomes detectable after treatment of fibroblasts with TGF by 24 h.

Cell Proliferation Genes. TGF regulated a cluster of genes involved in cell proliferation (Fig. 9D). We found upregulation of several genes, including CYR6 (IGFBP10), FGF2, IGF1, IGFBP7, PDGFA, and PTHLH. The induction of these genes was suppressed by SD-208, but not by SD-282. In addition, down-regulation of several antiproliferative genes, such as BTG1, CD164, and GAS1, was observed. The regulation of these genes was strongly suppressed by SD-208, and not by SD-282, except for GAS1, which showed modest reversal by SD-282. These data support a role for TGF in the positive regulation of cell proliferation in human lung fibroblasts after conversion to myofibroblasts in response to ligand-mediated signaling through TGFRI.

Apoptosis and Cell Survival Genes. TGF up-regulated antiapoptotic genes, including BAG3, BNIP1, DAD1, and IER3 (Fig. 9E). In addition, TGF down-regulated numerous proapoptotic genes, including CASP5, CED-6, CRADD, DAP, DAPK2, HTATIP2, PLAGL1, REQ, STK17A, TIA1, TFPT, and TNFSF10. All of these gene regulatory events were reversed by SD-208, whereas a subset, DAP, REQ, TNFSF10, and STK17B, was affected also by SD-282. These results suggest that TGF may protect myofibroblasts from apoptosis in human lung fibroblasts, and that TGF receptor blockage relieves this protection.

TGF and MAPK Pathway Genes. TGF was also found to regulate genes within the TGF and MAPK pathways (Fig. 9F and Supplemental Table E2). A majority of the genes were down-regulated by TGF, such as MADH1 (mothers against DPP homolog 1 or SMAD1), TGFBR3 (-glycan), MAP2K6 (MEK6), MAP3K5 (MEKK5), MAP4K4 (HGK), and MAPKAPK3 (3PK). We also observed TGF repression of SMAD3 (Table 3 and data not shown—one replicate at the 72 h TGF + SD-282 time point did not meet acceptable criteria as described under Materials and Methods). A few genes were up-regulated, including SKIL (SNO), MADH7 (SMAD7), and MAPK7 (ERK5). All TGF-induced gene expression changes in this group were reversed by SD-208. It is noteworthy that SD-282, the p38 MAPK inhibitor, also blocked TGF-down-regulation of MEK6 and MEKK5, which activate p38 and c-Jun NH₂-terminal kinase MAP kinases, respectively. Many of these gene changes represent novel TGF-induced events, such as the regulation of SNO, HGK, and 3PK. These results are consistent with ligand-induced feedback loops, which serve to regulate signaling mediated by SMADs and p38 MAPK.

Validation of Microarray by Real-Time RT-PCR and Western Analyses
Representative microarray data were validated in multiple HLF donors using real-time RT-PCR and Western analyses. The effects of TGF treatment and cotreatment with SD-208 or SD-282 were similar as measured by real-time RT-PCR and microarray for the tested genes: ACTA2, TIMP3, CTGF, IL11, PAI-1, COX2, IL6, SMAD3, SMAD7, IER3, PDGFA, fibronectin, and IGF1 (Table 3, three separate donors). SD-208 reversed the TGF effects of these genes, whereas SD-282 showed effects only on a subset of genes, including TIMP3, IL11, COX2, and fibronectin. Some differences between the microarray and real-time RT-PCR were noted for fibronectin. TGF-induction of fibronectin was inhibited similarly by SD-208 using these two methods, whereas partial inhibition by SD-282 was not detected by the differential expression threshold values of the microarray. Further validation of gene expression changes was observed at the protein level in two separate donors for CTGF, PAI-1, and -smooth muscle actin (Figs. 4 and 10, representative donor shown). Taken together, these results confirmed our microarray data using independent assay methods.

View larger version (25K): [in this window] [in a new window]   Fig. 10. Effects of SD-208 and SD-282 on TGF-induced PAI-1 and CTGF. Protein levels of PAI-1 (A and B) and CTGF (C-F) were measured by ELISA and Western blot analysis, respectively. Human lung fibroblast cells were treated with TGF in the presence of either SD-208 (400 nM) or SD-282 (400 nM) for 24 (A, C, and D) and 72 h (B, E, and F). The histograms in A-C and E represent mean data points from two separate experiments each including triplicate biological replicates (n = 6). The Western blot data are normalized to vimentin and expressed as a percentage compared with the TGF-treated samples. D and F are representative Western blots showing CTGF and Vimentin protein staining. ***, P =<0.001; **, P < 0.01 versus TGF.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Microarray analyses demonstrated that inhibition of TGFRI kinase activity by SD-208 opposed a majority (58-84%) of the TGF-induced gene expression changes from 7.5 to 72 h. In contrast, blockade of the p38 MAPK pathway by SD-282 affected a minor subset (10-18%) of these TGFmediated events during the same time course. TGFRI receptor blockade opposed TGF-regulated genes involved in fibrosis, myofibroblast transformation, cell proliferation, inflammation, and apoptosis. Inhibition of p38 MAPK demonstrated that a subset of the TGF-regulated events involved in inflammation and in fibrosis were mediated at least in part by p38.

Fibroblasts undergo transformation to myofibroblasts in response to TGF (Yokozeki et al., 1997). These smooth muscle-like cells are instrumental in growth, development, and repair of normal and diseased tissues. During normal wound repair, myofibroblast transformation and expression of ECM proteins are initiated in response to injury but terminate after healing when myofibroblasts disappear by apoptotic mechanisms (Desmouliere et al., 1995). In contrast, in fibrotic diseases such as pulmonary fibrosis, myofibroblasts persist, as evidenced by fibroblastic foci in diseased lungs (Nicholson et al., 2002), with continued proliferation and ECM expression. This dysregulated state is sustained by the expression of profibrotic and proinflammatory factors, including TGF, which act to prevent apoptosis and support continued expression of the factors by the myofibroblasts.

In the present study, we show that the process of myofibroblast transformation in human lung fibroblasts is dependent on signaling through the TGFRI and not the p38 pathway. The effects of inhibition of these pathways as they relate to myofibroblast conversion were measured by three independent methods: -smooth muscle actin protein staining, microarray gene expression of cytoskeletal and muscle contraction genes, and collagen gel contraction. Collagen gel contraction assays are accepted as functionally relevant as models of fibrotic tissue remodeling because contraction induced by TGF during wound healing is thought to depend, in part, on the promotion of myofibroblast differentiation (Lijnen et al., 2003a,b). We identified additional known markers (Manabe et al., 2002) of the conversion to myofibroblasts including MYH9, tropomyosin 1, and tropomyosin 4 through microarray analysis (Supplemental Table E2 and data not shown). Further supporting the transformation to the bona fide myofibroblast, we found several newly identified TGF-responsive genes that are involved in muscle contraction, such as caldesmon 1, calponin 1, calponin 3, dystrophin, and troponin C (Supplemental Table E2 and data not shown). Induction of these genes by TGF was reversed by SD-208 and not SD-282. The observation that the p38 path-way is not involved in myofibroblast conversion is consistent with previous reports (Hashimoto et al., 2001; Horowitz et al., 2004). Taken together, these data corroborate the role of TGF in myofibroblast conversion and show that SD-208 attenuates this fibrogenic process.

After activation of the myofibroblast by TGF, growth factors such as platelet-derived growth factor and CTGF, a member of the platelet-derived growth factor family, are thought to be responsible for myofibroblast proliferation (Powell et al., 1999). In the current study, we demonstrate that TGF induced the expression of PDGFA and CTGF, as well as several other growth factor genes (IGFBP10, FGF2, IGF1, and IGFBP7) implicated in cell proliferation. Moreover, we found that TGF down-regulated several novel responsive genes (BTG1, CD164, and GAS1) involved in anti-proliferative activities, further supporting its role in promoting myofibroblast proliferation. These TGF-regulated events were not affected by p38 inhibition but were opposed by SD-208. Thus, TGF's role in myofibroblast proliferation is two-fold: induction of proliferative growth factors and suppression of genes that normally act to inhibit cell proliferation; both of these actions were effectively blocked by SD-208.

There is extensive literature implicating myofibroblasts as key sources of fibrotic gene expression and production of ECM. Although not fully investigated, recent data suggest that bone marrow derived progenitor cells in the lung may also be an important source of collagen production in pulmonary fibrosis (Hashimoto et al., 2004). Here, we show that in HLF cells, TGF induced the expression of 20 genes involved in fibrogenic processes. Furthermore, we show that SD-208 globally opposes these fibrogenic gene changes. We found that a subset of the fibrotic genes (TIMP3, IL11, fibronectin) in myofibroblasts was regulated by both the TGF and p38 pathways. In most cases, the suppressive effects of SD-282 were modest compared with SD-208, suggesting that in a fibrogenic context, p38 pathway mediated events through TGFRI represent only partial signaling.

Myofibroblasts also play a major role in inflammatory responses (Phan et al., 1999). In the present study, we show that some of these effects are mediated by p38 signaling. We report for the first time that TGF up-regulation of several pro-inflammatory genes (IL1RAP, TNFAIP6, and TNFSF4) in HLF cells is mediated by the p38 pathway. We also observed that TGF induction of COX2, which is mediated in part by IL1RAP, is also opposed by SD-208 and SD-282. IL1RAP, interleukin 1 receptor accessory protein, forms a complex at the cell membrane with IL1 and its receptor, which is necessary for IL1 signal transduction. IL1 can mediate the induction of COX2 in intestinal fibroblasts (Mifflin et al., 2002) and the production of PGE2 in lung fibroblasts (Skold et al., 2000). Furthermore, we found that TGF downregulated several pro-inflammatory genes, such as MCP-4, MCP-3, MCP-2, and GRO1, consistent with previous reports showing that TGF plays a dual role in inflammatory processes (Kitamura, 1997; Gerritsma et al., 1998; Fong et al., 2000; Bradbury et al., 2002). It is noteworthy that the downregulation of these genes increased over time. These results raise the possibility that TGF may have a role in the resolution of the inflammatory phase of the myofibroblast. Other genes relating to inflammatory processes that are down-regulated by TGF include several members of the steroid receptor family (NR3C1/glucocorticoid receptor, NR2C1, NR2F1, and NR2F2) (Table E2). The down-regulation of these genes is relieved by TGFRI receptor blockade but not by p38 inhibition. Together, our results suggest that both TGF-induced SMAD signaling and p38 MAPK signaling are important in inflammatory responses in human lung fibroblasts. Moreover, the novel observation that effects of TGFRI blockade on steroid receptor genes raise the possibility that TGFRI inhibitors may be useful for increasing the efficacy of corticosteroids in inflammatory diseases such as asthma.

Successful wound repair mandates a process of myofibroblast elimination. This usually occurs after the proliferative and inflammatory phases subside. The mechanism of this resolution involves apoptosis (Desmouliere et al., 1995); however, the apoptotic process in the context of TGF signaling in myofibroblasts is not completely understood. Here, we show that TGF regulated numerous apoptotic genes; TGF up-regulated antiapoptotic genes and down-regulated proapoptotic genes. These gene groups also revealed several novel TGF-responsive genes such as CED-6, CRADD, DAP, DAPK2, HTATIP2, PLAGL1, REQ, and STK17A. In the context of extended TGF signaling, the gene changes suggest that TGF may serve to protect myofibroblasts from apoptosis. These data are consistent with a previous report showing that TGF protects myofibroblasts against IL-1-induced apoptosis (Zhang and Phan, 1999). SD-208 reversed the TGF-induced regulation of these apoptotic genes. The finding supports the notion that TGF-induced protection from apoptosis in human lung fibroblasts can be attenuated by TGFRI blockade and provides a rational for treating wounds susceptible to excessive scaring with inhibitors of TGFRI.

In summary, our study demonstrates that SD-208, the TGFRI inhibitor, but not SD-282, the p38 MAPK inhibitor, attenuated TGF-induced SMAD signaling and myofibroblast transformation in vivo and in vitro. Microarray analysis identified molecular fingerprints underlying these processes, and showed that SD-208 had global effects on reversing TGF-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Importantly, our studies support a therapeutic role for TGFRI inhibition in human interstitial lung diseases and support the use of inhibitors such as SD-208 for treating fibrotic conditions.

	Acknowledgements

 
We thank Lisa Garrard, Kip Madden, Jie Hu, and Estelle Tham for informatics expertise and support, all of which made this work possible. In addition, we are grateful to Carl Dowds and Evelyn Lawani for assistance in clone sequence verifications.

	Footnotes

 
N.J.G. and Y.W. contributed equally to this work

ABBREVIATIONS: ECM, extracellular matrix; TGF, transforming growth factor-; TGFR, transforming growth factor- receptor; MAPK, p38 mitogen-activated kinase; HLF, human lung fibroblast; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; FGM, fibroblast growth medium; BSA, bovine serum albumin; SMAD, mothers against DPP homolog; CTGF, connective tissue growth factor; PAI-1, plasminogen activator inhibitor, type I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PEG, polyethylene glycol.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Ann M. Kapoun, 6500 Paseo Padre Park-way, Fremont, CA 94555. E-mail: ann_kapoun{at}yahoo.com

	References

Top Abstract Materials and Methods Results Discussion References

 
Atfi A, Djelloul S, Chastre E, Davis R, and Gespach C (1997) Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor -mediated signaling. J Biol Chem 272: 1429-1432.[Abstract/Free Full Text]

Bakin AV, Rinehart C, Tomlinson AK, and Arteaga CL (2002) p38 mitogen-activated protein kinase is required for TGFbeta-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci 115: 3193-3206.[Abstract/Free Full Text]

Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL, and Arteaga CL (2000) Phosphatidylinositol 3-kinase function is required for transforming growth factor -mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 275: 36803-36810.[Abstract/Free Full Text]

Bell E, Ivarsson B, and Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76: 1274-1278.[Abstract/Free Full Text]

Bhowmick NA, Zent R, Ghiassi M, McDonnell M, and Moses HL (2001) Integrin beta 1 signaling is necessary for transforming growth factor- activation of p38MAPK and epithelial plasticity. J Biol Chem 276: 46707-46713.[Abstract/Free Full Text]

Bonniaud P, Margetts PJ, Kolb M, Schroeder JA, Kapoun AM, Damm D, Murphy A, Chakravarty S, Dugar S, Higgins L, et al. (2005) Progressive transforming growth factor {beta}1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Med 171: 889-898.[Abstract/Free Full Text]

Bradbury DA, Newton R, Zhu YM, Stocks J, Corbett L, Holland ED, Pang LH, and Knox AJ (2002) Effect of bradykinin, TGF-beta1, IL-1beta, and hypoxia on COX-2 expression in pulmonary artery smooth muscle cells. Am J Physiol 283: L717-L725.

Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jeffery PK, and McAnulty RJ (1997) Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. Am J Pathol 150: 981-991.[Abstract]

Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.[CrossRef][Medline]

Desmouliere A, Redard M, Darby I, and Gabbiani G (1995) Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 146: 56-66.[Abstract]

Fong CY, Pang L, Holland E, and Knox AJ (2000) TGF-beta1 stimulates IL-8 release, COX-2 expression, and PGE(2) release in human airway smooth muscle cells. Am J Physiol 279: L201-L207.

Frey RS and Mulder KM (1997) Involvement of extracellular signal-regulated kinase 2 and stress-activated protein kinase/Jun N-terminal kinase activation by transforming growth factor beta in the negative growth control of breast cancer cells. Cancer Res 57: 628-633.[Abstract/Free Full Text]

Gerritsma JS, van Kooten C, Gerritsen AF, van Es LA, and Daha MR (1998) Transforming growth factor-beta 1 regulates chemokine and complement production by human proximal tubular epithelial cells. Kidney Int 53: 609-616.[CrossRef][Medline]

Grande JP (1997) Role of transforming growth factor-beta in tissue injury and repair. Proc Soc Exp Biol Med 214: 27-40.[Abstract]

Green FH (2002) Overview of pulmonary fibrosis. Chest 122: 334S-339S.[Abstract/Free Full Text]

Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Investig 113: 243-252.[CrossRef][Medline]

Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Maruoka S, and Horie T (2001) Transforming growth Factor-beta1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway. Am J Respir Crit Care Med 163: 152-157.[Abstract/Free Full Text]

Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, and Gerthoffer WT (1999) A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 274: 24211-24219.[Abstract/Free Full Text]

Horowitz JC, Lee DY, Waghray M, Keshamouni VG, Thomas PE, Zhang H, Cui Z, and Thannickal VJ (2004) Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor. J Biol Chem 279: 1359-1367.[Abstract/Free Full Text]

Iwasaki S, Iguchi M, Watanabe K, Hoshino R, Tsujimoto M, and Kohno M (1999) Specific activation of the p38 mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein-2. J Biol Chem 274: 26503-26510.[Abstract/Free Full Text]<