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The Journal of Immunology, 2007, 179: 682-690.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Unc119 Regulates Myofibroblast Differentiation through the Activation of Fyn and the p38 MAPK Pathway1

Ramarao Vepachedu, Magdalena M. Gorska, Neil Singhania, Gregory P. Cosgrove, Kevin K. Brown and Rafeul Alam2

National Jewish Medical and Research Center and University of Colorado Health Sciences Center, Denver, CO 80206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of {alpha}-smooth muscle actin ({alpha}-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate {alpha}-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of {alpha}-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced {alpha}-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of {alpha}-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Myofibroblasts represent a potent source of tissue matrix proteins, inflammatory cytokines, and mediators in many chronic fibrotic and inflammatory conditions (1). Increased numbers of myofibroblasts have been observed in the airway tissue from asthmatic patients (2, 3). Myofibroblasts are considered to play a key role in collagen deposition and airway remodeling (4). The cellular origin of myofibroblasts and their mechanism of differentiation are not fully clear and require further studies. Various growth factors including TGF-beta and platelet-derived growth factor (PDGF)3 are potent inducers of myofibroblast differentiation in vitro (5, 6, 7). Their levels are elevated in the airway tissue from asthmatic patients (6, 8). The signal transduction mechanism of the foregoing growth factors frequently involves the activation of the Src family kinases (SFKs). The SFKs are involved in a wide variety of cellular functions including cell differentiation, motility, proliferation, and survival (9). In fibroblasts, Src, Fyn, and Yes associate with and are activated by the PDGFR (10, 11). Similarly, the TGF-betaR signal is partially mediated by the SFKs. In lung fibroblasts, the inhibition of the SFKs decreases the formation of {alpha}-smooth muscle actin ({alpha}-SMA) by TGF-beta (12). The formation of {alpha}-SMA is mediated though the SFK and focal adhesion kinase (9, 12). A kinase-deficient focal adhesion kinase, mutated by substituting Tyr397, inhibits TGF-beta-induced {alpha}-SMA expression and stress fiber formation.

The SFKs contain an N-terminal myristoylation site, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a linker region, and a kinase domain. The kinase is physiologically maintained inactive through multiple intramolecular interactions. One of these interactions occurs between the SH2 domain and the C-terminal phosphotyrosine residue. The other is between the SH3 domain and the linker region. Activation of the SFKs requires disruption of both intramolecular interactions (13), which leads to an open conformation and autophosphorylation of the activation loop. This stepwise process requires the involvement of a phosphatase and the interaction with a higher affinity SH3 ligand. We have recently cloned an adapter molecule called Unc119 that has multiple SH2- and SH3-binding motifs in addition to other protein-protein interaction motifs. Unc119 was cloned as an IL-5R{alpha}-interactive protein through a yeast two-hybrid screening of a hemopoietic cell cDNA library (14). We have shown that Unc119 interacts with the SFKs in eosinophils. Unc119 binds and activates Lyn and Hck kinases through its SH3- and SH2-binding motifs (14). Recently, we have shown that Unc119 plays a significant role in T cell signaling and is crucial for signal generation by the TCR (15). Unc119 functions as a receptor-associated activator of Lck and Fyn in T cells. In the present study, we examined the role of Unc119 in activation of the SFKs in fibroblasts and the differentiation of the latter into myofibroblasts by TGF-beta.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and medium

The mouse embryonic fibroblast (NIH3T3, CRL-1658), the human fetal lung fibroblast (IMR 190, CRL-186), and the SYF (embryonic fibroblast cell line with homozygous deletion of Src, Yes, and Fyn kinase; CRL-2459) cell lines were obtained from American Type Culture Collection (ATCC). Primary human lung and skin fibroblasts were obtained from the National Jewish Medical and Research Center Institutional Review Board-approved Interstitial Lung Disease Tissue Bank (16). Fibrotic lung fibroblasts were obtained as part of the National Heart, Lung, and Blood Institute (NHLBI)-sponsored interstitial lung disease Specialized Center of Research project. Control fibroblasts were obtained from single whole-lung samples (Tissue Transformation Technologies). All individuals suffered brain death and were evaluated for organ transplantation before research consent. Informed consent was obtained at the time of transplant evaluation. All specimens failed regional lung selection criteria for transplantation. The cells were maintained in medium consisting of DMEM (Mediatech) supplemented with 10% FCS (Cellgro) and antibiotics. The primary lung fibroblasts were supplemented with nonessential amino acids. Medium was changed every 3 days. Cells were plated on 35- or 100-mm dishes and incubated in 5% CO2 air at 37°C. For immunofluorescence studies, cells were plated on glass cover slips placed in 6-well plates.

Abs and fluorescence reagents

A rabbit polyclonal anti-Unc119 Ab was produced as described in Cen et al. (14). Abs against Fyn, Src, Yes, actin, phospho-p38, p38, phospho-JNK1, JNK1, pSmad2/3, and tubulin were obtained from Santa Cruz Biotechnology. The Ab against {alpha}-SMA was obtained from DakoCytomation. Alexa 488- and Alexa 594-labeled secondary Abs were obtained from Molecular Probes (Invitrogen Life Technologies).

Plasmids and Unc119 overexpression

Mouse and human Unc119 cDNAs were cloned into the bicistronic retroviral vector GFP-RV as described previously (15). The Unc119-GFP bicistronic retrovirus was produced by transfecting the Phoenix Ampho packaging cell line (ATCC) using TransFectin (Bio-Rad) as per the manufacturer’s protocol. The supernatants were collected 48 h after transfection and sterilized with 0.45-µm filters. Target cells were overlaid with viral supernatants mixed 1:1 with the growth medium (DMEM plus 10% FCS) and 8 µg/ml polybrene and then centrifuged in plates at 1200 x g for 90 min at 32°C. The cells expressing Unc119 were selected at 48 h by sorting the GFP-expressing cells. GFP-Unc119 fusion protein was constructed in the pLEGFP-N1 vector (BD Clontech). Mouse Fyn cDNA cloned in pCMV-SPORT6 vector was obtained from Open Biosystems. For Unc119-GFP fusion protein expression, the NIH3T3 cells were transfected with the plasmid using TransFectin (Bio-Rad) and stable transfectants were selected by growing the cells in the presence of G418. For transient transfection studies, cells were transfected with the Amaxa Nucelofector kit.

Immunoprecipitation and Western blotting

For the immunoprecipitation, cells were lysed in the lysis buffer containing 50 mM Tris-HCl (pH 7.4), 75 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 0.5% Nonidet P-40, 1 µg/ml each of the protease inhibitors aprotinin, leupeptin, and pepstatin, and 1 mM PMSF, at a density of 2 x 106 cell/100 µl of the lysis buffer. The cell lysate was incubated on ice for 30 min and detergent-insoluble materials were removed by centrifugation at 4°C at 13,000 x g. The lysates were precleared with 20 µl of protein A/G agarose/100 µl of lysate for 1 h. An appropriate Ab (2 µg) was added to the lysate and incubated at 4°C rotating for 1 h or overnight followed by addition of 20 µl of protein A/G agarose. The incubation was continued for 2 h or overnight. The mixture was centrifuged at 12,000 x g for 5 min and the pellet was washed three times with 1 ml of lysis buffer and three times with the kinase buffer in the case of a kinase assay. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for immunoblotting. The membranes were incubated in 5% BSA in TBST buffer for 1 h followed by incubation in the primary Ab at a concentration of 0.2 µg/ml in 5% BSA/TBST buffer. The membranes were then washed three times in the TBST buffer for 10 min each and incubated with a HRP-conjugated secondary Ab (Santa Cruz Biotechnology) solution. After additional washing, the membranes were developed with the ECL (ECL Plus) substrate. To strip and reprobe, the membranes were incubated in the stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl (pH 6.7)) at 55°C for 30 min, washed, blocked, and immunoblotted with an appropriate Ab as described above. Densitometric analyses of select Western-blotted protein bands were performed with the software "ImageJ" (http://rsb.info.nih.gov/ij/).

Immunostaining and fluorescent microscopy

For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% saponin in TBST. The samples were blocked with 10% goat serum for 1 h at room temperature and then incubated with primary Abs followed by corresponding labeled secondary (goat) Abs. The cells were examined under a Nikon 2000 Epifluorescence microscope fitted with a Coolsnap camera. The pictures were captured, processed, and the fluorescence intensity was measured using the Metamorph software (Molecular Devices).

Unc119-deficient cells

Small interfering RNA (siRNA) for Unc119 and control were introduced into NIH3T3 cells in 6-well plates using the transfecting reagent Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. siRNA and lipofectamine complexes were made with 200 pM siRNA and 5 µl of Lipofectamine 2000 in 250 µl of Opti-MEM I medium (Invitrogen Life Technologies) separately and then mixing them together. The complexes were allowed to form at room temperature for 20 min and then plated on 30–50% confluent cells in 6-well plates. The medium was changed after 16 h. For transfecting IMR90 cells, siRNA was introduced by the Nucleofector kit (Amaxa) as per the manufacturer’s protocol. The suppression of Unc119 expression was confirmed by Western blotting. For generation of Unc119-deficient cells and siRNA control cells, siGENOME SMARTpool reagent for mouse Unc119 and siControl NonTargeting siRNA were obtained from Dharmacon. The identification numbers for mouse and human siUnc119 and siControl are M-063215-00-0010 and M-009734-00-0005.

Cell activation and {alpha}-SMA production

The Unc119-overexpressing or -deficient IMR90 human lung fibroblasts were made quiescent by reducing the concentration of FCS in the medium to 0.01% for 24 h before stimulation with TGF-beta (1 ng/ml). Porcine platelet-derived TGF-beta was obtained from R&D Systems. The cells were harvested at different time points and equal amounts of the cellular protein extract were used to study {alpha}-SMA expression.

In vitro kinase assays

To measure the activation of the SFKs in control and Unc119-overexpressing or Unc119-knockdown fibroblasts, the kinases were immunoprecipitated with respective Abs (Santa Cruz Biotechnology) from the cell lysate (2 million cells/assay). An autophosphorylation assay was performed in a kinase buffer containing 20 mM Tris (pH 7.4), 2 mM MgCl2, 0.5 µM cold ATP, and 2 µCi [{gamma}-32P]ATP (Amersham Biosciences) for 15 min. For substrate phosphorylation, 2 µM enolase (Sigma-Aldrich) was added to the reaction buffer. For studies of Fyn activation by TGF-beta and PDGF-BB in the presence and absence of Unc119, cells were transfected with control and Unc119 siRNA. Cells were starved to quiescence for 24 h in 0.01% FBS in DMEM in 6-well plates. The medium was changed with fresh DMEM containing TGF-beta or PDGF-BB. The cells were incubated at 37°C in a 5% CO2 chamber. The medium was removed and the cells were harvested at indicated time points by lysing with 1x radioimmunoprecipitation assay (RIPA) buffer followed by kinase assay. The kinase reaction was stopped by addition 4x Laemmli’s buffer. The samples were then separated on 12% polyacrylamide gels (SDS-PAGE), transferred to a polyvinylidene difluoride membrane, and autoradiographed. For quantitation of the kinase activity the membranes were exposed to a storage phosphor screen (Amersham Biosciences) and the activity was measured using the Typhoon Variable Mode Imager. The quantity was estimated by ImageQuant software (Amersham Biosciences).

Proliferation assay

NIH3T3 cells (10,000) were cultured for 24 h in a 99-well plate and then [3H]thymidine (Amersham Biosciences) was added at 1 µCi/well. Cells were harvested 24 h later and [3H] radiation was measured by a liquid scintillation counter.

Detection of cytokines

IMR90 cells expressing Unc119 or control vector and IMR90 cells transfected with siUnc119 or siControl RNA were seeded in a 12-well plate and cultured in DMEM containing 10% FBS. The cells were washed twice with PBS at 24 h and then cultured in DMEM alone without FBS. Cell-free medium was collected at 24 and 48 h. Cell viability was >80% at the conclusion of the culture. The cell-free medium was assayed for basal cytokine production by the SerachLight Testing Service (Pierce Biotechnology). Samples from three independent experiments were assayed in duplicate for the following cytokines: IL-1beta, IL-4, IL-6, IL-11, IL-13, TNF-{alpha}, epidermal growth factor (EGF), and TGF-beta.

Data presentation

All experiments were repeated at least three to five times. The results from multiple experiments were presented as the mean ± SE or as a representative result from three or more independent experiments. Statistical analyses were done by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Unc119 regulates TGF-beta-induced expression of {alpha}-SMA

In addition to their physiological role in tissue homeostasis, fibroblasts play an important role in pathologic conditions by differentiating into myofibroblasts. TGF-beta is one of many factors that induce this differentiation. A characteristic feature of myofibroblast differentiation is the expression of {alpha}-SMA. We examined the effect of Unc119 on {alpha}-SMA expression in human fetal lung fibroblasts (IMR90) using Unc119-overexpression and -knockdown models. We overexpressed Unc119 in lung fibroblasts using the bicistronic retroviral vector GFP-RV (GFP-expressing retrovirus). The cells expressing GFP were sorted and used for experiments. The overexpression of Unc119 was confirmed by Western blotting. The presence of higher levels of Unc119 increased the induction of {alpha}-SMA. In Unc119-overexpressing cells, the stimulation with 1 ng of TGF-beta resulted in an overall increase in {alpha}-SMA compared with mock vector-transfected cells (Fig. 1A). At 48 h, {alpha}-SMA induction was increased by 2-fold. The increased induction of {alpha}-SMA in the presence of Unc119 was confirmed by immunostaining. The cells overexpressing Unc119 showed an increase in {alpha}-SMA staining (Fig. 1B). We also examined whether the increased smooth muscle actin expression in Unc119-overexpressing cells was mediated through the phosphorylation of Smad2/3. We detected baseline phosphorylation of Smad2/3 in both control and Unc119-overexpressing cells likely due to the effect of the growth factors that are present in the serum of the culture medium (Fig. 1B). The distribution of pSmad2/3 was mostly cytoplasmic under these conditions. Stimulation with TGF-beta caused nuclear translocation of pSmad2/3. However, there was no difference in cytoplasmic or nuclear pSmad2/3 between control and Unc119-overexpressing cells. The results suggest that Unc119 does not directly increase the phosphorylation of Smad2/3 or its nuclear translocation.


Figure 1
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  		FIGURE 1. Unc119 influences {alpha}-SMA formation in lung fibroblasts. A, Vector control and Unc119-overexpressing lung fibroblasts (IMR190) cells were starved for 24 h and then activated with TGF-beta1 (1 ng/ml). The cells were lysed at indicated time points and the proteins were resolved by SDS-PAGE. The expression of {alpha}-SMA was studied by Western blotting. The overexpression of Unc119 was demonstrated by Western blotting with an anti-Unc119 Ab after stripping the blot (note the upper band in the blot is a nonspecific band). The blot was next reprobed for equal protein loading by Western blotting for actin and pSmad2/3 (n = 3). B, {alpha}-SMA expression in lung fibroblasts. The cells were grown as mentioned above on glass cover slips. At indicated time points, the cells were fixed in 4% paraformaldehyde, incubated with an anti-{alpha}-SMA Ab, followed by an Alexa 594-conjugated secondary Ab (n = 3) (left panel). Scale bar, 5 µm. The integrated fluorescence intensity was measured using Metamorph software. The amount of {alpha}-SMA in control at the 0 time point was used as a unit to measure the expression of {alpha}-SMA. Values from five different fields from each experiment were used (right panel). {alpha}-SMA expression between RV and Unc119 is statistically significant (p < 0.05, t test). C, Distribution of pSmad2/3. Cells stained for pSmad2/3 localization at 0 and 48 h time points. Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI) (n = 3).

 
Unc119 deficiency lowers TGF-beta-induced {alpha}-SMA production

To further confirm that Unc119 modulates the expression of {alpha}-SMA, we examined its induction in lung fibroblasts using Unc119-knockdown cells. In Unc119 siRNA-treated cells, the expression of Unc119 was 27% of the control (Fig. 2A). In these Unc119-deficient cells, the TGF-beta-induced expression of {alpha}-SMA was decreased (Fig. 2B) compared with control nontargeting siRNA-treated cells. There was a 40% reduction in {alpha}-SMA in the absence of Unc119. Thus, results from Unc119-deficient and -overexpressing cells indicate that Unc119 regulates {alpha}-SMA induction by TGF-beta.


Figure 2
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  		FIGURE 2. Unc119 affects {alpha}-SMA expression. A, Expression of Unc119. Lung fibroblasts (IMR90) were transfected with siRNA targeting Unc119 (siUnc119) and siControl (SMART pool; Dharmacon). The cells were harvested after 48 h and the expression of Unc119 was examined by Western blotting (upper panel). The blot was checked for equal protein loading by Western blotting for beta-tubulin (lower panel). B, Unc119 deficiency inhibits {alpha}-SMA expression. The lung fibroblasts were transfected with siUnc119 and siControl RNA. The next day, the cells were starved for 24 h and then activated with TGF beta1. The expression of {alpha}-SMA was studied by Western blotting. The blot was checked for equal protein loading by Western blotting for actin. Lower panel, The quantitation of {alpha}-SMA expression (pixel density). Data are presented as fold increase over siControl (n = 4). The difference in pixel density at 24 and 48 h between siUnc119 and siControl is statistically significant (p < 0.001, t test).

 
Unc119 overexpression activates Fyn

The induction of {alpha}-SMA has previously been shown to be facilitated by the SFKs (12). Unc119 activates certain members of the SFKs through its SH2- and SH3-binding motifs (14). Fibroblasts express three members of SFKs: Src, Yes, and Fyn. To study which of these kinases is activated by Unc119, we examined their activation state in Unc119-overexpressing cells. These studies were performed using the mouse embryonic fibroblast cell line NIH3T3 because of the availability of the SYF cell line, a mouse embryonic fibroblast cell line with homozygous null mutation for the Src family kinases Src, Yes, and Fyn. The SYF cell line will allow us to determine the role of the SFKs in Unc119 function and myofibroblast differentiation in a definitive manner. No such triple kinase-deficient lung fibroblast cell lines are available. Unc119 was overexpressed in NIH3T3 fibroblasts using the bicistronic retroviral vector GFP-RV. The overexpression of Unc119 was confirmed by Western blotting (Fig. 3A). From the same cell extracts, the Src family kinases Src, Yes, and Fyn were studied for their autophosphorylation in an immune complex kinase assay in the presence of radiolabeled ATP. The baseline activity of the SFKs remained the same except for Fyn indicating the activity of Src and Yes kinases was not affected by Unc119 overexpression (Fig. 3B). The kinase activity of Fyn was up-regulated by overexpression of Unc119. The Fyn activity showed a 72% up-regulation in Unc119-overexpressing cells over the control RV-infected cells (Fig. 3C).


Figure 3
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  		FIGURE 3. Unc119 overexpression activates Fyn in fibroblasts. A, NIH3T3 cells were transfected with Unc-GFP-RV or GFP-RV. Following sorting of GFP-positive cells, cell lysates were Western blotted with an anti-Unc119 Ab (upper panel) followed by reprobing with beta-tubulin for equal protein loading (lower panel). B, Autophosphorylation of Src, Yes, and Fyn. Overnight, grown control and Unc119-overexpressing cells were lysed and the SFKs were immunoprecipitated with respective Abs (rabbit). The autophosphorylation (incorporation of 32P) was examined in a kinase assay. The blot was reprobed with respective anti-SFK Abs. Representative phosphorylation from three independent kinase assays is shown. C, The incorporation of 32P in the kinase autophosphorylation assay was measured using a phosphoimager and the results of three experiments are shown in the bar diagrams as a percent of control (RV). Fyn activation was significantly (p < 0.03) increased in Unc119-overexpressing cells. D, Immunoprecipitation of Unc119 with SFKs. The SFKs were immunoprecipitated with the respective Abs and Western blotted for coprecipitation of Unc119. Equal amount of rabbit IgG was used for mock immunoprecipitation (IP). E, Colocalization of Unc119 with Fyn in fibroblasts. Cell lines expressing GFP and Unc119-GFP fusion protein, obtained by stable transfection of NIH3T3 cells were grown in the presence of serum overnight on cover slips. Cells were fixed and immunostained with an anti-Fyn Ab followed by treatment with an Alexa 596-labeled secondary Ab (n = 3). The arrow indicates the area of colocalization that was magnified in the inset. Top right panel, Immunofluorescence staining pattern of endogenous Unc119 in NIH3T3 cells. Bottom right panel, The Ab control. Cells were prepared and fixed as above and then incubated with a rabbit anti-Unc119 Ab or control rabbit serum followed by Alexa 596-labeled secondary Ab. A representative image is shown (n = 3).

 
Unc119 directly interacts with the SFKs through the SH3 domain for kinase activation (14). We asked whether Fyn and other SFKs physically interacted with Unc119 in fibroblasts. Immunoprecipitation studies showed that only Fyn, but not Src or Yes, interacted with Unc119 (Fig. 3D). We examined colocalization of Fyn with Unc119. To this goal, we used NIH3T3 cells stably expressing the GFP-Unc119 fusion protein. The expression pattern of GFP-Unc119 was similar to that of endogenous Unc119 (Fig. 3E). Colocalization studies with the GFP-Unc119 fusion protein showed a small but consistent colocalization of Unc119 with Fyn (Fig. 3E). GFP alone did not colocalize with Unc119. GFP-Unc119 did not colocalize with other Src kinases (data not presented).

Unc119 deficiency delays Fyn activation by TGF-beta and PDGF-BB

Many growth factor receptors use the SFKs to generate intracellular signals. TGF-beta and PDGF-BB are major growth factors for fibroblasts. Like TGF-beta, PDGF-BB also causes myofibroblast differentiation (7). One of the downstream effectors of the PDGFR is Fyn. We examined the activation of Fyn by TGF-beta and PDGF-BB in Unc119-knockdown cells. Fyn autophosphorylation was significantly reduced in Unc119-deficient cells at early time points (15 and 30 min; Fig. 4, A and B). Fyn autophosphorylation in control cells reached the peak by 30 min after TGF-beta and PDGF-BB treatment. Under similar conditions, Unc119 deficiency resulted in only 20% autophosphorylation of Fyn. However, by 60 min, Fyn autophosphorylation in Unc119-deficient cells was similar to that seen in control cells. The kinase activity of Fyn was measured by enolase (substrate) phosphorylation. The substrate phosphorylation results were concordant with the autophosphorylation results. These data indicate that Unc119 is important for the early phase activation of Fyn by the TGF-beta and PDGF receptors.


Figure 4
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  		FIGURE 4. Unc119 required for SFK activation. A and B, Effect of Unc119 knockdown on TGF-beta (A) and PDGF-BB (B) induced Fyn autophosphorylation. After transfecting with control and Unc119 siRNA, NIH3T3 fibroblasts were allowed to grow for a day and then starved for 24 h with 0.01% FBS followed by activation with TGF-beta (2 ng/ml) or PDGF-BB (20 µg/ml) for various lengths of time. The cells were lysed with the radioimmunoprecipitation assay buffer and immunoprecipitated with an anti-Fyn rabbit Ab. The immunoprecipitates were used in the autophosphorylation and substrate (enolase) phosphorylation assays. The blots were reprobed with a mouse anti-Fyn Ab for equal protein loading (n = 3). The substrate (enolase) phosphorylation (incorporation of radioactive 32P) was measured using a phosphoimager and presented as a bar diagram (side panel). C, Unc119 deficiency causes decreased cell proliferation. NIH3T3 cells and SYF cells were transfected with siControl and siUnc119 as described under A and cultured in DMEM with 10% FBS. After 24 h, the cells were transferred to a 96-well plate and [3H]thymidine (Amersham Biosciences) was added at 1 µCi/well. Cells were harvested 24 h after the addition of [3H]thymidine and the incorporation of [3H] was measured by a gamma counter. The results represent mean of three independent experiments.

 
Unc119 regulates fibroblast proliferation through SFK

To understand its physiological importance, we studied the effect of Unc119 knockdown on fibroblast proliferation. Serum-stimulated proliferation of NIH3T3 fibroblasts in the absence of Unc119 was studied by the [3H]thymidine incorporation assay. Proliferation was only 48% in Unc119-knockdown cells when compared with siControl cells (Fig. 4D, p < 0.001). The inhibition of thymidine incorporation indicates an essential role for Unc119 in fibroblast proliferation. To explore the role of other signaling pathways in Unc119 regulation of fibroblast proliferation, we took advantage of the SYF cell line. As mentioned earlier, this cell line has null mutation for all three fibroblast-associated SFKs: Src, Yes, and Fyn. Basal proliferation rate of this cell line is ~25% slower than that of the control embryonic fibroblast cell line (NIH3T3). Serum-induced [3H]thymidine incorporation of SYF and control cells were 211 and 269 x 103 dpm, respectively. Unc119 knockdown did not significantly affect thymidine incorporation in SYF cells. These experiments suggest that the effect of Unc119 on proliferation is primarily mediated by the SFKs.

Unc119 modulates {alpha}-SMA induction through Fyn

The SFKs are involved in a variety of biological responses that are elicited by TGF-beta. Because Unc119 activates Fyn in fibroblasts, we examined its role in {alpha}-SMA expression using the SYF fibroblast cell line. In the absence of the SFKs, the expression of {alpha}-SMA by TGF-beta was very low in SYF cells (Fig. 5A). Unc119 overexpression was unable to overcome this defective induction of {alpha}-SMA. Our results suggest that the SFKs are important for the expression of {alpha}-SMA and that Unc119 exerts its regulatory influence on {alpha}-SMA through the SFKs. To confirm the involvement of Fyn in {alpha}-SMA production, the SYF cells were transfected with an Fyn expression vector and the expression of {alpha}-SMA was checked. The reconstitution resulted in normal expression of {alpha}-SMA confirming the requirement of Fyn for {alpha}-SMA synthesis (Fig. 5B). Double transfection with the Unc119 and Fyn vectors resulted in enhanced expression of {alpha}-SMA as compared with the Fyn vector alone. These results confirm that the enhanced {alpha}-SMA synthesis in presence of Unc119 is mediated through Fyn.


Figure 5
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  		FIGURE 5. {alpha}-SMA synthesis requires SFK. A, SFK deficiency inhibits {alpha}-SMA expression. The SYF cells were transfected with Unc119-GFP-RV and GFP-RV bicistronic vectors. The efficiency of transfection was checked by the expression of GFP, which was >70% at 24 h. The cells were starved for a day and then stimulated with TGF-beta (1 ng/ml) as described for lung fibroblasts under Fig. 1A. The expression of {alpha}-SMA was studied by Western blotting and the protein loading was examined by reprobing the membrane for actin (n = 3). B, Fyn expression restores synthesis of {alpha}-SMA. The SYF cells were transiently cotransfected with a Fyn expression vector along with Unc119-GFP-RV or GFP-RV vector. The cells were stimulated with TGF-beta as above and then Western blotted for {alpha}-SMA (n = 3).

 
Unc119 regulates TGF-beta-induced expression of {alpha}-SMA through p38 MAPK

The major signaling pathway in TGF-beta signaling involves the Smad family of transcription factors. Other signaling molecules that have been implicated in the TGF-beta-signaling pathway include RhoA, PKN, JNK, and p38 MAPK. We studied the activation of p38 MAPK by TGF-beta in IMR90 lung fibroblasts. The activation of p38 MAPK reached the peak in 2 h and then returned to baseline by 6 h (Fig. 6A). There was no further activation at 24 and 48 h. The activation of p38 MAPK can be mediated by the SFKs. To address this question, we stimulated cells with TGF-beta in the presence of PP2, an SFK inhibitor. The activation of p38 MAPK was inhibited in these experiments confirming the importance of the SFKs in p38 activation. Then, we studied the activation of p38 MAPK and JNK by TGF-beta in Unc119-overexpressing cells. The control cells showed no activation of p38 MAPK after the addition of TGF-beta at 24 or 48 h as shown in Fig. 6A. However, in Unc119-overexpressing cells, the p38 MAPK showed increased phosphorylation at 24 and 48 h (Fig. 6B). Thus, Unc119 facilitates sustained activation of p38 MAPK in TGF-beta-stimulated cells. In contrast to p38, JNK did not show any increase in phosphorylation in Unc119-overexpressing cells. These results suggest that activation of p38 MAPK could play a role in the induction of {alpha}-SMA synthesis in Unc119-overexpressing cells. To further explore this issue, the expression of {alpha}-SMA was investigated in the presence of the p38 MAPK inhibitor SB202193. The expression of {alpha}-SMA in Unc119-overexpressing cells was blocked by SB202193 (Fig. 6C). The results suggest that p38 MAPK is involved in the Unc119-mediated expression of {alpha}-SMA.


Figure 6
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  		FIGURE 6. Unc119 enhances {alpha}-SMA synthesis by SFK and p38 MAPK. A, SFK mediates the activation of p38 MAPK by TGF-beta. IMR90 cells were starved for 24 h and then activated with TGF-beta (1 ng/ml) in absence (upper panel) or presence (lower panel) of PP2. The cells were lysed at the indicated time points and checked for phosphorylation of p38 MAPK by Western blotting. The membrane was reprobed with an anti-p38 Ab (n = 3). B, Unc119 enhances the activation of p38 MAPK. Control and Unc119-overexpressing cells were stimulated with as described in Fig. 1A and then phosphorylation of p38 and JNK1 were examined at 24 and 48 h. The blots were reprobed for total p38 and JNK1 (lower panels) (n = 4). C, The inhibition of p38 MAPK suppresses {alpha}-SMA expression. Control and Unc119-overexpressing cells were treated with TGF-beta in presence and absence of the p38 inhibitor SB212090 (10 µM). The expression of {alpha}-SMA was studied by Western blotting (n = 3).

 
Unc119 expression results in increased IL-6 and TGF-beta production

The biological outcome of Unc119 overexpression could depend on the secretion of cytokines apart from the intracellular signal transduction events. Cytokine production could secondarily enhance {alpha}-SMA synthesis. To investigate this possibility, we measured basal production of cytokines in Unc119-overexpressing and Unc119-deficient cells cultured short-term in the absence of serum. The avoidance of serum was important because it contains low quantities of many cytokines and growth factors. We studied the secretion of IL-1beta, IL-4, IL-6, IL-11, IL-13, TNF-{alpha}, EGF, and TGF-beta. These cytokines were chosen because of their association with myofibroblast differentiation and function. Overexpression of Unc119 resulted in increased expression of IL-6 and TGF-beta (Fig. 7, A and B). In control cells, IL-6 production was low, 55 ± 7 pg/ml at 24 h and did not change at 48 h. However, in Unc119-overexpressing cells, the expression was high: 414 ± 220 pg/ml at 24 h and 1784 ± 376 pg/ml at 48 h. Similarly, fibroblasts produced low levels of TGF-beta under basal conditions, which did not change over time (191 ± 15 and 196 ± 21 pg of TGF-beta/ml at 24 and 48 h, respectively). But in Unc119-overexpressing cells, the secretion of TGF-beta increased from 166 ± 21 pg/ml at 24 h to 415 ± 96 pg/ml at 48 h. The secretion of these cytokines was further studied in Unc119-deficient cells (Fig. 7, C and D). In control siRNA and siUnc119-treated cells, the secretion of IL-6 decreased with time. IL-6 production decreased from 55 ± 7 pg/ml at 24 h to 28 ± 3 pg/ml at 48 h in control cells and from 61 ± 13 pg/ml at 24 h to 10 ± 6 pg/ml at 48 h in siUnc119 cells. TGF-beta secretion was similar at 24 and 48 h in control cells (~200 pg/ml). However, siUnc119-treated cells showed significantly reduced TGF-beta secretion (110 ± 28 pg/ml, p < 0.01) at 48 h. EGF was undetectable in our samples. IL-1beta, IL-4, IL-11, IL-13, and TNF-{alpha} did not show any significant changes upon Unc119 overexpression and knockdown.


Figure 7
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  		FIGURE 7. Unc119 increases the production of IL6 and TGF-beta. A and B, IMR90 cells overexpressing Unc119 or control vector were seeded in a 12-well plate and cultured in DMEM containing 10% FBS. The cells were washed twice with PBS at 24 h and then cultured in DMEM alone without FBS. Cell-free medium was collected at 24 and 48 h. Cell viability was >80% at the conclusion of the culture. The cell-free medium was assayed for cytokine production by the "Searchlight" (Pierce) multiplex assay (n = 3). The results for IL-6 and TGF-beta are shown. C and D, IMR90 cells were transfected with siControl and siUnc119 RNA and then cultured as described above. Cell-free medium was collected at 24 and 48 h. The secretion of cytokines was measured by the "Searchlight" multiplex assay. The effect of Unc119 knockdown on IL-6 and TGF-beta secretion is shown (n = 3). *, p < 0.001.

 
Primary lung fibroblasts from patients with fibrotic lung diseases show {alpha}-SMA and Unc119 coexpression

Myofibroblasts play a seminal role in the pathogenesis of pulmonary fibrosis. We studied the expression of {alpha}-SMA and Unc119 in human lung fibroblasts from three patients with fibrotic lung diseases and three nondiseased control subjects (Table I). In addition, we used human dermal fibroblasts (donor 4, Table I). Primary fibroblasts were grown on cover slips for 3 days with and without TGF-beta as described previously and stained for {alpha}-SMA and Unc119. Fibroblasts from all study subjects showed baseline expression of {alpha}-SMA (data not shown). TGF-beta stimulation further increased this expression. We compared the TGF-beta-induced expression of {alpha}-SMA among various donors (Fig. 8A). From the standpoint of the fibroblast origin, dermal fibroblasts showed the lowest expression of both {alpha}-SMA and Unc119. Specimens from two patients and two control subjects showed strong and comparable levels of {alpha}-SMA expression (Fig. 8B). Fibroblasts from the third subject from both groups showed moderate levels of {alpha}-SMA expression. More importantly, the expression of Unc119 paralleled that of {alpha}-SMA. We performed morphometric analyses of integrated fluorescence intensity of Unc119 and {alpha}-SMA immunostaining from randomly selected three images obtained from each study subject (Fig. 8B). There was a significant correlation (r = 0.79, Pearson’s correlation coefficient) between Unc119 and {alpha}-SMA expression in these images. In agreement with previously published reports (16), fibroblasts from the fibrotic lungs did not express higher levels of {alpha}-SMA under in vitro conditions when compared with that from the nondiseased lungs. A line-scan analysis of individual cells from the images taken from the same field shows a remarkable overlap of expression peaks. The cells with the highest Unc119 level also expressed the highest level of {alpha}-SMA (Fig. 8C).


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  		Table I. Source of lung fibroblast cell lines used

 

Figure 8
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  		FIGURE 8. {alpha}-SMA and Unc119 coexpression. Unc119 expression is associated with {alpha}-SMA in the primary fibroblasts obtained from lung specimens. A, Primary lung fibroblasts from three pulmonary fibrosis patients and three nondiseased lung donors and dermal fibroblasts from a healthy donor were grown for 3 days on coverslips in the presence of TGF-beta and then fixed with paraformaldehyde. The cells were immunostained for Unc119 and {alpha}-SMA. Representative images from dermal fibroblasts and lung fibroblasts obtained from a control subject and from a pulmonary fibrosis patient are shown. Scale bar, 20 µm. B, A scatter plot of integrated fluorescence intensity of three randomly selected paired (Unc119 and {alpha}-SMA) images from three fibrotic patients and three control donors (NDF: normal dermal fibroblasts; NLF: normal lung fibroblasts; FLF: fibrotic lung fibroblasts). C, Lung fibroblasts from a pulmonary fibrosis patient was transfected with Unc119 and control siRNA. The cells were allowed to grow 48 h under low FBS condition (0.01%) and the cell lysates were immunoblotted for {alpha}-SMA. Lower panel, Equal loading by reprobing the membrane for actin. D, A line scan of fluorescence intensity of Unc119 (green) and {alpha}-SMA (red) staining across a single image (left panel) of fibrotic lung fibroblasts.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Unc119 is a ubiquitously expressed protein. Unc119 was identified as a neural gene and found to be involved in feeding, chemosensation, and locomotion in Caenorhabditis elegans (17, 18). Unc119 suppresses axon branching in C. elegans and is required to stabilize the differentiated structure of the nervous system (19). Recent studies indicate that Unc119 plays an important role in the development and function of the vertebrate nervous system. Morpholino oligonucleotide-mediated knockdown of Unc119 protein in zebra fish results in missing or misplaced cell bodies, defasciculation, axon pathfinding errors, and aberrant axonal branching (20). In transgenic mouse, expression of the mutated Unc119 (HRG4) under rhodopsin promoter caused retinal degeneration (21, 22). Although its biological function in the CNS, especially in retina, has been well-characterized, its role in many other cell types has not been examined.

Previous studies from our group have shown that Unc119 activates Lck and Fyn in T cells, and Lyn and Hck in eosinophils (14, 15). The foregoing findings raised the possibility that Unc119 might activate other members of SFKs. Among the three SFKs expressed by fibroblasts, we show that Unc119 activates only Fyn but not Src or Yes. Unc119 contains two separate SH3-binding motifs and one SH2-binding motif. The SH3 motifs are predicted to bind to the SH3 domain of SFKs. Immunoprecipitation studies have shown that Unc119 associates with Fyn, but not Src or Yes. Colocalization studies confirm this observation. This supports the view that a physical interaction facilitates the activation of Fyn. The lack of activation of Src and Yes by Unc119 may suggest microheterogeneity within the consensus binding sequence or different intracellular distribution.

The SFKs are known to associate with the activated receptor for PDGF, EGF, CSF, nerve growth factor, and hepatocyte growth factor (10, 23, 24, 25). The SFKs are substrates for activated FGFR (26) and PDGFR (11). The catalytic activation of Fyn by the TGF-beta and PDGF receptors was delayed in the absence of Unc119 in our studies. Thus, Unc119 plays an essential role in the early phase activation of the SFKs associated with TGF-beta and PDGF receptors. The loss of Fyn activation in Unc119-deficient cells appears to have functional consequences. Knockdown of Unc119 significantly inhibits cell proliferation. This effect of Unc119 is likely mediated by Fyn, which has previously been shown to be important for fibroblast proliferation (26). In the absence of the SFKs in SYF cells, the knockdown of Unc119 does not affect proliferation. In zebra fish, the knockdown of Unc119 results in many developmental disorders such as missing cell bodies, defasciculation, axon path-finding errors, and aberrant axonal branching (21). However, the pathways involved in such aberration are not known. It is of interest that Fyn deficiency causes developmental abnormalities, especially defective myelination in the CNS in mice (27). Our results indicate that Unc119 deficiency results in impaired Fyn activation and defective cell proliferation.

Using fibroblasts as model cells, we show that Unc119 regulates myofibroblast differentiation. The differentiation of fibroblasts into myofibroblasts with high expression of {alpha}-SMA has been observed in wound healing and in various pathological conditions such as asthma and pulmonary fibrosis (28, 29, 30, 31). TGF-beta plays a crucial role in myofibroblast differentiation (32, 33, 34, 35). It signals through Smad-dependent and -independent pathways (36). Unc119 modulates the expression of {alpha}-SMA induced by TGF-beta. We have observed that the overexpression of Unc119 results in heightened production of {alpha}-SMA. Conversely, in the absence of Unc119, the production of {alpha}-SMA was reduced. Thus, Unc119 plays an important role in {alpha}-SMA induction.

To further confirm that the SFKs are essential for myofibroblast differentiation, we have studied the effect of TGF-beta stimulation of SYF cells. Cells deficient in the SFKs have a profound defect in {alpha}-SMA expression. This defect could not be overcome by overexpressing Unc119 indicating the Unc119 effect is mediated by the SFKs. Expression of Fyn in SYF cells restored the ability to synthesize {alpha}-SMA. Under these reconstituted conditions, expression of Unc119 further enhanced the productions of {alpha}-SMA. These results clearly prove that {alpha}-SMA synthesis is mediated by the SFKs and that the enhancement of {alpha}-SMA synthesis by Unc119 requires, at least, the presence of Fyn. We studied the biological relevance of Unc119 for myofibroblast differentiation using primary lung fibroblasts from patients with fibrotic lung diseases. We observed a significant correlation between Unc119 expression and {alpha}-SMA production in myofibroblasts obtained from the lung specimens. It needs to be determined whether a similar coexpression occurs in situ in the lung tissue from patients with fibrotic lung diseases.

It is well-established that TGF-beta induces myofibroblast differentiation. The mechanism of signal transduction of TGF-beta leading to myofibroblast differentiation is not completely understood. Recent studies have suggested that the TGF-beta/MAPK/AP-1 pathway regulates {alpha}-SMA synthesis in human fetal lung fibroblasts (36). Both ERK and p38MAPK were involved in the synthesis of {alpha}-SMA in the above study. In human lung fibroblasts, TGF-beta stimulates p38 MAPK activation rapidly within 30 min, which returns to baseline by 2 h (34). In human tendon fibroblasts, the activation of p38 MAPK appears to be biphasic with second activation occurring at 24 h. In the present study, p38 MAPK is phosphorylated in the first 2 h following TGF-beta stimulation of lung fibroblasts. PP2 abrogates this activation confirming that p38 activation depends on SFK. Unc119 overexpression results in sustained activation of p38 MAPK, which remains phosphorylated at 24 and 48 h. The activation of p38 is important because its inhibition substantially reduces {alpha}-SMA expression. A molecular complex of phosphorylated Smad2, Smad3, and Smad4 makes an optimal transcriptosome for induction of fibrogenic genes. TGF-betaRI phosphorylates Smad2. In TGF-beta-stimulated cells, Smad3 undergoes phosphorylation at two different sites—one in the linker region (Ser207/212) and the other in the C-terminal region (Ser423/425). It has been recently shown that phosphorylation in the linker region is mediated by the p38 MAPK (37). We speculate that this increased p38 MAPK activation leads to Smad3 phosphorylation in Unc119-overexpressing cells. The convergence of Smad signaling and the SFK signaling results in heightened {alpha}-SMA synthesis. It should be pointed out that the blocking of p38 pathway reduces overall expression of {alpha}-SMA expression regardless of Unc119 expression. This means the TGF-beta-induced expression of {alpha}-SMA is partially mediated through the Unc199-p38 MAPK pathway.

Cytokines influence inflammation and myofibroblast differentiation. IL-6 and TGF-beta play an important role in chronic inflammatory conditions. In addition to TGF-beta, IL-6 has recently been reported to augment myofibroblast differentiation and {alpha}-SMA induction in dermal fibroblasts during wound healing (38). Unc119 up-regulates the expression of both IL-6 and TGF-beta. Interestingly, IL-6 and TGF-beta were shown to have an autocrine loop through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells (39). The levels of TGF-beta that are induced by Unc119 are insufficient to stimulate {alpha}-SMA production. We believe that TGF-beta and IL-6, working in concert, exert a synergistic effect. Our results suggest that Unc119 modulates fibroblast function not only through the modification of signaling pathways, but also through the regulation of cytokine secretion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants RO1 AI50179, AI059719, and AI68088. Back

2 Address correspondence and reprint requests to Dr. Rafeul Alam, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: alamr{at}njrc.org Back

3 Abbreviations used in this paper: PDGF, platelet-derived growth factor; SFK, Src family kinase; {alpha}-SMA, {alpha}-smooth muscle actin; RV, retroviral vector; SH, Src homology; EGF, epidermal growth factor; siRNA, small interfering RNA. Back

Received for publication August 25, 2006. Accepted for publication April 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Powell, D. W., R. C. Mifflin, J. D. Valentich, S. E. Crowe, J. I. Saada, A. B. West. 1999. Myofibroblasts. I. Paracrine cells important in health and disease. Am. J. Physiol. 277: C1-C9. [Medline]
  2. Brewster, C. E., P. H. Howarth, R. Djukanovic, J. Wilson, S. T. Holgate, W. R. Roche. 1990. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3: 507-511. [Medline]
  3. Gizycki, M. J., E. Adelroth, A. V. Rogers, P. M. O’Byrne, P. K. Jeffery. 1997. Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am. J. Respir. Cell Mol. Biol. 16: 664-673. [Abstract]
  4. Elias, J. A., Z. Zhu, G. Chupp, R. J. Homer. 1999. Airway remodeling in asthma. J. Clin. Invest. 104: 1001-1006. [Medline]
  5. Varga, J., J. Rosenbloom, S. A. Jimenez. 1987. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem. J. 247: 597-604. [Medline]
  6. Batra, V., A. I. Musani, A. T. Hastie, S. Khurana, K. A. Carpenter, J. G. Zangrilli, S. P. Peters. 2004. Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on {alpha}-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts. Clin. Exp. Allergy 34: 437-444. [Medline]
  7. Sommer, M., U. Eismann, J. Gerth, and G. Stein. 2002. Interleukin 4 co-stimulates the PDGF-BB- and bFGF-mediated proliferation of mesangial cells and myofibroblasts. Nephron 92: 868–880; discussion 880–862.
  8. Redington, A. E., J. Madden, A. J. Frew, R. Djukanovic, W. R. Roche, S. T. Holgate, P. H. Howarth. 1997. Transforming growth factor-beta1 in asthma: measurement in bronchoalveolar lavage fluid. Am. J. Respir. Crit. Care Med. 156: 642-647. [Abstract/Free Full Text]
  9. Thomas, S. M., J. S. Brugge. 1997. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13: 513-609. [Medline]
  10. Twamley-Stein, G. M., R. Pepperkok, W. Ansorge, S. A. Courtneidge. 1993. The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells. Proc. Natl. Acad. Sci. USA 90: 7696-7700. [Abstract/Free Full Text]
  11. Hansen, K., G. Alonso, S. A. Courtneidge, L. Ronnstrand, C. H. Heldin. 1997. PDGF-induced phosphorylation of Tyr28 in the N-terminus of Fyn affects Fyn activation. Biochem. Biophys. Res. Commun. 241: 355-362. [Medline]
  12. Thannickal, V. J., D. Y. Lee, E. S. White, Z. Cui, J. M. Larios, R. Chacon, J. C. Horowitz, R. M. Day, P. E. Thomas. 2003. Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J. Biol. Chem. 278: 12384-12389. [Abstract/Free Full Text]
  13. Sicheri, F., J. Kuriyan. 1997. Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7: 777-785. [Medline]
  14. Cen, O., M. M. Gorska, S. J. Stafford, S. Sur, R. Alam. 2003. Identification of UNC119 as a novel activator of SRC-type tyrosine kinases. J. Biol. Chem. 278: 8837-8845. [Abstract/Free Full Text]
  15. Gorska, M. M., S. J. Stafford, O. Cen, S. Sur, R. Alam. 2004. Unc119, a novel activator of Lck/Fyn, is essential for T cell activation. J. Exp. Med. 199: 369-379. [Abstract/Free Full Text]
  16. Frankel, S. K., G. P. Cosgrove, S. I. Cha, C. D. Cool, M. W. Wynes, B. L. Edelman, K. K. Brown, D. W. Riches. 2006. TNF-{alpha} sensitizes normal and fibrotic human lung fibroblasts to Fas-induced apoptosis. Am. J. Respir. Cell Mol. Biol. 34: 293-304. [Abstract/Free Full Text]
  17. Maduro, M., D. Pilgrim. 1995. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141: 977-988. [Abstract]
  18. Maduro, M. F., M. Gordon, R. Jacobs, D. B. Pilgrim. 2000. The UNC-119 family of neural proteins is functionally conserved between humans, Drosophila and C. elegans. J. Neurogenet. 13: 191-212. [Medline]
  19. Knobel, K. M., W. S. Davis, E. M. Jorgensen, M. J. Bastiani. 2001. UNC-119 suppresses axon branching in C. elegans. Development 128: 4079-4092. [Medline]
  20. Manning, A. G., B. D. Crawford, A. J. Waskiewicz, D. B. Pilgrim. 2004. unc-119 homolog required for normal development of the zebrafish nervous system. Genesis 40: 223-230. [Medline]
  21. Higashide, T., G. Inana. 1999. Characterization of the gene for HRG4 (UNC119), a novel photoreceptor synaptic protein homologous to unc-119. Genomics 57: 446-450. [Medline]
  22. Kobayashi, A., T. Higashide, D. Hamasaki, S. Kubota, H. Sakuma, W. An, T. Fujimaki, M. J. McLaren, R. G. Weleber, G. Inana. 2000. HRG4 (UNC119) mutation found in cone-rod dystrophy causes retinal degeneration in a transgenic model. Invest. Ophthalmol. Vis. Sci. 41: 3268-3277. [Abstract/Free Full Text]
  23. Zhan, X., C. Plourde, X. Hu, R. Friesel, T. Maciag. 1994. Association of fibroblast growth factor receptor-1 with c-Src correlates with association between c-Src and cortactin. J. Biol. Chem. 269: 20221-20224. [Abstract/Free Full Text]
  24. Kypta, R. M., Y. Goldberg, E. T. Ulug, S. A. Courtneidge. 1990. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell 62: 481-492. [Medline]
  25. Abram, C. L., S. A. Courtneidge. 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell Res. 254: 1-13. [Medline]
  26. Kilkenny, D. M., J. V. Rocheleau, J. Price, M. B. Reich, G. G. Miller. 2003. c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts. J. Biol. Chem. 278: 17448-17454. [Abstract/Free Full Text]
  27. Sperber, B. R., E. A. Boyle-Walsh, M. J. Engleka, P. Gadue, A. C. Peterson, P. L. Stein, S. S. Scherer, F. A. McMorris. 2001. A unique role for Fyn in CNS myelination. J. Neurosci. 21: 2039-2047. [Abstract/Free Full Text]
  28. Phan, S. H.. 2002. The myofibroblast in pulmonary fibrosis. Chest 122: 286S-289S. [Medline]
  29. Pardo, A., M. Selman. 2002. Idiopathic pulmonary fibrosis: new insights in its pathogenesis. Int. J. Biochem. Cell Biol. 34: 1534-1538. [Medline]
  30. Redington, A. E.. 2000. Fibrosis and airway remodelling. Clin. Exp. Allergy 30: (Suppl. 1):42-45. [Medline]
  31. Gabbiani, G.. 2003. The myofibroblast in wound healing and fibrocontractive diseases. J. Pathol. 200: 500-503. [Medline]
  32. Ina, K., H. Kitamura, S. Tatsukawa, T. Takayama, Y. Fujikura, T. Shimada. 2002. Transformation of interstitial fibroblasts and tubulointerstitial fibrosis in diabetic nephropathy. Med. Electron Microsc. 35: 87-95. [Medline]
  33. Kissin, E. Y., J. H. Korn. 2003. Fibrosis in scleroderma. Rheum. Dis. Clin. North Am. 29: 351-369. [Medline]
  34. Hashimoto, S., Y. Gon, I. Takeshita, K. Matsumoto, S. Maruoka, T. Horie. 2001. Transforming growth factor-beta1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway. Am. Respir. Crit. Care Med. 163: 152-157. [Abstract/Free Full Text]
  35. Javelaud, D., A. Mauviel. 2005. Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta: implications for carcinogenesis. Oncogene 24: 5742-5750. [Medline]
  36. Hu, Y., J. Peng, D. Feng, L. Chu, X. Li, Z. Jin, Z. Lin, Q. Zeng. 2006. Role of extracellular signal-regulated kinase, p38 kinase, and activator protein-1 in transforming growth factor-beta1-induced {alpha} smooth muscle actin expression in human fetal lung fibroblasts in vitro. Lung 184: 33-42. [Medline]
  37. Furukawa, F., K. Matsuzaki, S. Mori, Y. Tahashi, K. Yoshida, Y. Sugano, H. Yamagata, M. Matsushita, T. Seki, Y. Inagaki, et al 2003. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology 38: 879-889. [Medline]
  38. Gallucci, R. M., E. G. Lee, J. J. Tomasek. 2006. IL-6 modulates {alpha}-smooth muscle actin expression in dermal fibroblasts from IL-6-deficient mice. J. Invest. Dermatol. 126: 561-568. [Medline]
  39. Aoki, H., H. Ohnishi, K. Hama, S. Shinozaki, H. Kita, H. Yamamoto, H. Osawa, K. Sato, K. Tamada, K. Sugano. 2006. Existence of autocrine loop between interleukin-6 and transforming growth factor-beta1 in activated rat pancreatic stellate cells. J. Cell. Biochem. 99: 221-228. [Medline]




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