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GROWTH, DIFFERENTIATION, AND APOPTOSIS
-regulated collagen type I accumulation: role of Src-based signals1Division of Nephrology and Hypertension, Department of Medicine, and 2Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio
Submitted 7 July 2006 ; accepted in final form 21 November 2006
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ABSTRACT |
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(TGF-) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF--induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF--treated cells than in controls. Src activation by TGF- was blocked by rottlerin and by a dominant negative mutant of protein kinase C
(PKC), showing that TGF- activates Src by a PKC-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF- requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF- requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF- requires a PKC-Src-ERK1/2 signaling motif.
mesangial cells; fibrosis; glomerulus; transforming growth factor-
(TGF-) stimulates myofibroblast transdifferentiation, contributing to fibrosis and end-stage organ failure in the lung, liver, heart, and kidney (47, 48). TGF- induces myofibroblast transdifferentiation by autocrine or paracrine signaling from inflammatory, epithelial, or tumor cells. In vitro, TGF- directs the diverse phenotypic changes of myofibroblast transdifferentiation: cell growth; 
-smooth muscle actin expression; and accumulation of extracellular matrix, particularly fibronectin and interstitial collagen types I and III (6, 48). In vivo, adenoviral transfer of TGF- in rat lung evokes myofibroblast transdifferentiation and chronic fibrosis (40). Similarly, targeting bioactive TGF- to the mouse lung provokes myofibroblast transdifferentiation and fibrosis (20). Although other ligands are involved (i.e., endothelin-1, connective tissue growth factor), TGF- is a crucial regulator of myofibroblast transdifferentiation and fibrosis.
In vitro and in vivo, TGF--Smad signaling is required for myofibroblast transdifferentiation (16, 23, 25, 37, 47, 48), particularly for accumulation of extracellular matrix, but TGF- signaling is modulated by kinases to confer maximal and cell type-specific responses (5, 23, 30). In some cases TGF- slowly activates kinases, suggesting that these might be delayed, indirect effects, but in some cells TGF- rapidly activates kinases, consistent with a direct mechanism of action. For instance, TGF- receptors activate extracellular signal-regulated kinases 1/2 (ERK1/2), which phosphorylate regulatory Smads and, depending on cell context, exert positive or negative effects on Smad-dependent transcription (24). Experiments with Smad 4-deficient cells, with dominant negative Smads, or with mutant TGF- type I receptors defective in Smad activation suggest that TGF- can activate the c-Jun NH2-terminal kinase (JNK) and p38 MAPK independently of Smads (10, 49). Non-Smad signaling can proceed by mechanisms that require direct binding of alternative effectors, such as protein phosphatase PP2A, to the TGF- receptor (30). Therefore, although Smads lie at the core of TGF- signaling, downstream signaling from TGF- receptors also involves kinases that are highly dependent on cellular context.
Few firm data exist on the role of nonreceptor protein tyrosine kinases in myofibroblast transdifferentiation. TGF- activates (19, 35, 45) or inhibits (1, 11, 21) the Src nonreceptor tyrosine kinase, which regulates cell growth and survival by TGF-. However, whether TGF--Src signals regulate myofibroblast transdifferentiation is unknown. Therefore, we investigated the role of Src in TGF--stimulated collagen type I accumulation in glomerular mesangial cells, a well-established in vitro model of myofibroblast transdifferentiation (9, 13, 17). We report that Src is required for collagen type I accumulation in human mesangial cells (HMC) exposed to TGF-.
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MATERIALS AND METHODS |
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-actin (Sigma Chemical, St. Louis, MO). PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, a cell-permeable pharmacological inhibitor of Src, its inactive analog PP3, and SU 6656 (3), originally developed at SUGEN, were obtained from Sigma. Bisindolylmaleimide I (BIS), Gö-6976, and rottlerin were obtained from (EMD Biosciences, San Diego, CA). Recombinant human TGF-1 was obtained from R&D Systems (Minneapolis, MN), and human endothelin-1 (ET-1) was obtained from American Peptides (Sunnyvale, CA).
Human mesangial cell culture.
HMC obtained from Cambrex Bioscience (Walkersville, MD) were cultured in Dulbecco's modified essential medium (DMEM; GIBCO-BRL 11885) supplemented with 17% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 5 ng/ml selenite, and 5 µg/ml each of insulin and transferrin in a CO2 incubator at 37°C in 5% CO2. Cells were used in passages 4–10. Characterization was performed by phase-contrast microscopy and by immunostaining for intermediate filaments and surface antigens (28, 39). HMC were positive for desmin, vimentin, and myosin but did not stain for factor VIII, keratin, or common leukocyte antigen. Before stimulation with TGF- or ET-1, HMC in 60-mm plates at 
80% confluence were made quiescent for 24 h in DMEM with 0.5% FBS.
Measurements of secreted and cell-associated collagen.
Cell-associated type I collagen was measured by Western blotting or direct ELISA. Secreted type I collagen was measured by ELISA. For Western blotting of cell-associated collagen type I, lysates were prepared as described below. The blots were probed with a polyclonal anti-human collagen type I antibody (1:1,000) with minimal cross-reactivity to other human collagens. To quantify secreted and cell-associated collagen type I, we developed an direct ELISA specific for human collagen type I. Quiescent HMC treated with TGF- were coincubated with 50 µg/ml -aminopropionitrile to minimize cross-linking. Supernatants were collected and immediately frozen; cell-associated collagen type I was solubilized in a 5 M guanidine-0.1 M Tris buffer (pH 8.6) with protease inhibitors as described previously (51). One hundred microliters of the medium, cell extract, or human collagen type I standards (Southern Biotechnology) were absorbed to Nunc Maxisorp 96-well plates overnight at 4°C. After three washes with PBS-0.1% Tween, nonspecific binding was blocked by 1.0% BSA in 100 mM phosphate buffer, pH 8.2. The wells were then incubated with a rabbit anti-human collagen type I, an affinity-purified and biotin-conjugated goat anti-rabbit IgG, a horseradish peroxidase-conjugated streptavidin, and a mix of 3,5,2',5'-tetramethylbenzizine, followed by quenching with acid and an absorbance reading at 450 nm (all secondary reagents were obtained from Jackson Immunoresearch). The plates were extensively washed between each step. Unknowns were predicted from a log-log fit of the standard curve using SoftMax Pro 4.6 (Molecular Devices), and all values were in the linear range of the standard curve and were normalized for cell number.
Measurement of steady-state collagen type I mRNA.
Real-time RT-PCR was used to measure collagen I mRNA. Briefly, total RNA was isolated (RNeasy; Qiagen, Valencia, CA), and 5 µg were used to synthesize cDNA with a T7-(dT)24 primer (Genset, La Jolla, CA) and RT Superscript II (GIBCO-BRL, Rockville, MD) for 1 h at 42°C. Primers for human Col1A1 mRNA (GenBank accession no. NM_000088) were designed using Primer3 (available at http://frodo.wi.mit.edu). Sequences of the primers for Col1A1 were as follows: upstream, GTG CTA AAG GTG CCA ATG GT; downstream, ACC AGG TTC ACC GCT GTT AC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control. Primers for GAPDH were as follows: upstream, TGT CCC CAC TGC CAA CGT GT; downstream, AGG GTA CTT TAT TGA TGG TA. Real-time PCR was carried out using a Stratagene MX3000P machine for 40 cycles as follows: 30 s at 95°C, 30 s at 68°C, and 30 s at 72°C, preceded by 10 min of incubation at 95°C. Using agarose gel electrophoresis of the PCR products, we confirmed that only one band of the predicted molecular weight was present. A melting curve recorded at the end of the reaction was used for correction of the amplification curve. The amount of mRNA in TGF--stimulated samples was calculated as described previously (26).
Western blotting for phospho-Src and phospho-ERK1/2.
Analysis of phosphoproteins in HMC by Western blotting was performed as previously described (27, 29) with minor modifications. Briefly, monolayers washed with ice-cold PBS were scraped into CHAPS extraction buffer [50 mM PIPES-HCl, pH 6.5, 2 mM EDTA, 0.1% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 5 mM DTT, 2 mM Na-pyrophosphate, 1 mM Na3VO4, and 1 mM NaF]. An aliquot was used for protein determination using the DC assay (Bio-Rad, Hercules, CA). An equal volume of 2x SDS sample buffer was immediately added to the extraction buffer, and Western blotting was carried out using antibodies for phospho-Src or phospho-ERK1/2. Membranes were reprobed for total Src, ERK2, or -actin to confirm equal protein loading. As previously described (26), the Western blots were analyzed by densitometry in NIH Image by normalizing values for the relevant protein to the control, untreated sample within each experiment.
siRNA-mediated Src gene knockdown by RNA interference.
We recently described the protocol for siRNA Src knockdown in HMC (29). The same pool of four complementary siRNA oligonucleotide duplexes targeted specifically to human Src (GenBank accession no. NM_005417) were used in these experiments (Dharmacon, Lafayette, CO). siRNAs were transfected into mesangial cells with Lipofectamine 2000 (Invitrogen). Quiescent HMC at 50–70% density in 35-mm wells were transfected with siRNA duplexes at 20 nM (29) in 2 ml of low serum-containing OPTI-MEMI (5% FBS) for 18 h. After incubation with siRNAs in low-serum medium, the concentration of serum was adjusted to that in complete medium and cells were grown for 48 h. Total Src was assessed by Western blotting to confirm Src siRNA knockdown. In parallel, non-target siRNA (Dharmacon; catalog no. D-001210) at 20 nM was used to control for off-target changes in mesangial cells. HMC in low-serum medium were then stimulated with TGF-, and collagen type I accumulation was measured.
Adenoviral transduction of HMC with dominant negative PKC and Src.
Replication-defective adenoviral vectors encoding dominant negative PKC (Ad5-dnPKC), dominant negative Src (Ad.KI-Src), and control (empty) adenovirus (Ad5-EV) were previously described (7, 8, 34). The dominant negative PKC mutant has a Lys-to-Arg mutation in the ATP-binding site (33), and it is transcribed from a composite CAG promoter with a cytomegalovirus immediate-early enhancer, chicken -actin promoter, and rabbit -globin polyadenylation signal. Kinase-dead, dominant negative Src has a mutation in the conserved lysine located in the Src kinase domain (34). Dominant negative Src was a generous gift of Drs. Bradford Berk and Kathy Griendling. All constructs were amplified in 293 cells and purified by cesium chloride ultracentrifugation. Viral titers were determined as plaque-forming units. For transduction, HMC were incubated with Ad5-dnPKC or Ad5-EV at a multiplicity of infection of 50 for 12 h. The optimal multiplicity of adenoviral infection was determined using a green fluorescent protein-encoding adenovirus. Under these conditions, the transduction efficiency was >90%.
Data analysis and statistics. Data are means ± SD from 3–4 independent experiments. Statistical significance was calculated using unpaired Student's t-test or ANOVA with Bonferroni multiple correction as appropriate (32), using InStat 3 (GraphPad).
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RESULTS |
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rapidly activates Src in HMC.
Using quiescent HMC, an in vitro model of myofibroblast transdifferentiation, we first asked whether TGF- activates Src. As an index of Src activity, immunoreactive PTyr416 Src was measured by Western blotting. PTyr416 Src was rapidly elevated by TGF- (Fig. 1A). Total Src protein was unchanged. The kinetics of Src activation by TGF- were similar to activation by ET-1 (Fig. 1, B and C), an established stimulus of Src in HMC (29, 41). In HMC exposed to TGF-, PTyr416 Src was elevated at 10 min, was highest at 20 min, and fell back toward basal level at 60 min (Fig. 1, A and C). At 20 min, PTyr416 Src was 3.3-fold higher in cells exposed to TGF- than in quiescent mesangial cells. These results show that TGF- rapidly stimulates Src in HMC.
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activates Src by a pathway requiring PKC.
We next investigated how TGF- activates Src in HMC. To determine whether PKC was involved, we pretreated quiescent HMC with BIS, a broad spectrum pharmacological antagonist of PKCs. BIS blocked the rise in PTyr416 Src in HMC treated with TGF- (Fig. 2, A and B). Blockade of Src activation by BIS argues that conventional or novel PKCs contribute to the TGF- signaling pathway that activates Src.
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activates Src, we pretreated HMC with Gö-6976, a potent and selective inhibitor of the Ca2+-dependent conventional PKCs that does not inhibit kinase activity of the Ca2+-independent novel PKCs even at micromolar levels (22). When added to quiescent HMC, Gö-6976 did not inhibit the rise in PTyr416 Src in cells exposed to TGF- (Fig. 2, C and D). Thus it appeared unlikely that conventional or atypical PKCs mediate Src activation by TGF-. Because the results with BIS and Gö-6076 suggested that a novel PKC was involved, we employed rottlerin, a selective pharmacological inhibitor of PKC (12). In the presence of rottlerin, TGF- no longer stimulated Src activity (Fig. 2, C and D). Our results suggest that TGF- activates Src by a mechanism involving PKC.
Although the PKC inhibitors used in the experiments described above are selective and were used at minimally efficacious doses, we cannot rule out possible non-PKC effects of these drugs. We therefore employed a loss-of-function, dominant negative mutant of PKC to test the role of PKC in TGF--Src signaling in HMC. HMC were transduced with adenovirus-expressing dominant negative PKC, made quiescent, and then stimulated with TGF-. Transduction with Ad5-dnPKC blocked the increase in Src activity in cells treated with TGF- (Fig. 3, A and B). These results support the hypothesis that TGF- activates Src in HMC by a PKC-based mechanism.
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-treated HMC requires Src.
Accumulation of collagen type I is a hallmark of myofibroblast transdifferentiation, and accumulation of collagen type I results in fibrosis, so we next asked whether Src participates in the signaling pathway by which TGF- elevates collagen type I accumulation in HMC. To measure collagen accumulation by TGF-, we first assessed type I collagen in HMC monolayers by Western blotting with an antibody specific for human type I collagen. TGF- increased type I procollagen (Fig. 4A). Over the same time course -actin was unchanged, showing that cell protein was equivalent in each lane. In additional experiments we used ELISA to measure secretion of type I collagen into the medium of HMC exposed to TGF-. Secretion of collagen type I was higher in cells treated with TGF- than in cells treated with medium alone (Fig. 4B). Following 24 h of exposure to TGF-, type I collagen secretion increased 2.1-fold (Fig. 4B). In addition, TGF- increased cell-associated type I collagen as measured by ELISA (Fig. 4C). As expected in myofibroblast transdifferentiation, these results demonstrate that TGF- increases collagen type I.
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-regulated type I collagen accumulation, we treated HMC with pharmacological inhibitors of Src, PP2, and SU 6656. At concentrations that selectively inhibit Src, pretreatment with PP2 blocked collagen type I secretion and deposition in cells exposed to TGF- for 48 h (Fig. 5, A and B). Another stimulus of myofibroblast transdifferentiation, ET-1, also increased collagen type I levels, and the increment in collagen was blocked by PP2 (Fig. 5, A and B). Forty-eight hours after TGF- or ET-1 treatment, collagen type I was 2.4- and 2.1-fold higher, respectively. The inactive but chemically similar analog PP3 had no effect on collagen type I accumulation (Fig. 5, A and B). SU 6656 also prevented the increment in collagen type I deposition and secretion in cells exposed to TGF- (Fig. 5, C and D). To confirm the requirement for Src, we measured secretion and deposition of collagen type I in cells transduced with an adenoviral vector expressing dominant negative Src (Fig. 5, C and D). Kinase-dead Src blocked the increase in collagen type I secretion and deposition in cells stimulated with TGF-. A vector lacking the Src insert did not affect collagen type I deposition or secretion, demonstrating that the effect of dominant negative Src was not a nonspecific effect of adenoviral transduction in mesangial cells (Fig. 5, C and D). We also showed that PP2, SU 6656, and dominant negative Src prevented induction of collagen type I mRNA in TGF--treated cells (Fig. 5E). Together, these results support a role for Src in TGF--mediated collagen type I induction.
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-induced collagen type I accumulation, we knocked down Src with siRNA, using a previously published protocol (29). We transfected mesangial cells for 48 h with 20 nM on-target Src siRNA and off-target siRNA. We have previously established that 20 nM is the lowest effective concentration for the Src siRNAs in HMC (29). Western blotting for total Src confirmed that on-target Src siRNAs blocked expression of Src protein, whereas the off-target complexes had no effect on Src (Fig. 6A). HMC were rendered quiescent in low-serum medium before addition of TGF- and measurement of cell-associated type I collagen by Western blotting. On-target Src siRNAs prevented the increase in collagen type I protein in HMC exposed to TGF- (Fig. 6, B and C). In contrast, TGF--induced collagen type I was unaffected by the off-target siRNAs (Fig. 6, B and C). The reduction in collagen type I by on-target Src siRNAs was reproducibly observed in three independent experiments (Fig. 6C). We note that -actin was unaffected by on- or off-target siRNAs, which suggests that the siRNA concentrations used in these experiments did not induce an interferon-like response that blocks protein synthesis. Therefore, knocking down Src protein prevents collagen type I accumulation in HMC exposed to TGF- and supports the results with PP2 antagonism of Src.
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. Because we also determined that TGF- activates Src by a mechanism involving PKC, we hypothesized that PKC also would be needed to increase collagen type I in HMC exposed to TGF-. Transduction with dominant negative PKC inhibited secreted and cell-associated type I collagen in HMC treated with TGF- (Fig. 7, A and B). Transduction of HMC with the adenoviral vector lacking an insert had no effect on either secretion or cell-association of type I collagen. These data demonstrate that a PKC-Src signaling pathway is necessary to increase collagen type I in HMC exposed to TGF-.
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-Src signaling cassette activates ERK1/2.
We next investigated the downstream effectors by which TGF--Src signaling increases collagen type I. Schnaper and colleagues (14, 38) have shown in HMC that, besides Smads, induction of collagen type I by TGF- requires ERK1/2 activity (14, 38). We have previously demonstrated that ERK1/2 is downstream of a Src-Ras signaling motif in HMC (15, 42). Src can regulate ERKs in several cell types (2). We therefore reasoned that activation of ERK1/2 by TGF- might proceed by a Src-dependent mechanism. When added to quiescent HMC, TGF-1 rapidly increased ERK1/2 phosphorylation, a readout of ERK1/2 kinase activity (Fig. 8, A and B). Preincubation with a dose of PP2 shown to block Src activity in HMC (29) and with SU 6656 greatly attenuated the increase in ERK1/2 activity in HMC exposed to TGF- (Fig. 8, A and B). Dominant negative Src, but not the empty vector, also prevented the increase in ERK1/2 activity in cells exposed to TGF-. Together with previous experimental results in HMC showing a requisite role for ERK1/2 in TGF- signaling to the collagen type I gene (14, 38), our present results demonstrate that Src is upstream of ERK1/2 in the TGF- signaling pathway that stimulates collagen type I accumulation in HMC.
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activates ERK1/2 by a Src-dependent mechanism, and because we showed that TGF- activates Src by a PKC-based pathway (Fig. 2 and 3), we asked whether activation of ERK1/2 by TGF- also requires PKC. TGF- rapidly raised ERK1/2 activity (Fig. 9, A and B), which was unaffected by Gö-6976, but ERK1/2 activity in cells exposed to TGF- was inhibited by rottlerin, the selective inhibitor of PKC. Activation of ERK1/2 by TGF- also was prevented by dominant negative PKC (Fig. 9B). Together with the results presented above, these experiments establish that a TGF--PKC-Src signaling pathway activates ERK1/2 in HMC.
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DISCUSSION |
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signaling through Smad proteins is widely recognized to initiate myofibroblast transdifferentiation and drive fibrosis in the lung, heart, liver, and kidney. However, myofibroblast transdifferentiation in cells exposed to TGF- also requires less well-understood kinase interactions. In this study we investigated the role of Src in an important aspect of TGF--stimulated HMC, namely, secretion and deposition of the interstitial collagen type I. We report that Src is necessary for collagen type I secretion and deposition in HMC exposed to TGF-. Furthermore, we demonstrate a Src ERK1/2-independent pathway that raises collagen type I secretion and a Src ERK1/2-dependent pathway that raises cell-associated type I collagen.
TGF- activates Src in mesangial cells.
We were prompted to investigate whether Src regulates myofibroblast transdifferentiation by our finding that TGF- activates Src in HMC. TGF- previously has been shown to increase (19, 35, 45) or decrease (1, 11, 21) Src activity. Cell type-specific differences in TGF- signaling have been proposed to explain these apparently conflicting results, but the dependence of TGF--Src signaling on cell context has not been directly tested. Moreover, the phenotypic responses regulated by TGF--Src in these cell types have not been extensively characterized. In our current studies with HMC, TGF- rapidly raised PTyr416 Src, a marker of Src activity. The increase in PTyr416 Src was maximal at 20 min and was returning toward baseline at 60 min. This robust activation of Src by TGF- was similar to the increase in Src activity by ET-1. This comparison is important, because ET-1 is a well-characterized activator of Src in mesangial cells (41, 42), and Src activity is required for induction of the c-fos immediate-early gene (15, 42), cyclin D1 (29), and progression through the cell cycle (29). Also, the rapid time course of Src activation by TGF- suggested a direct mechanism of action rather than an indirect effect that calls for de novo gene and protein expression.
TGF- activates Src by a PKC-based pathway.
As a first step in determining how TGF- activates Src, we investigated a possible role for PKC. PKC proteins belong to a large family of serine-threonine kinases that includes PKA and PKG. The PKC family consists of three major subtypes: conventional PKCs that need diacylglycerol, Ca2+, and phospholipids for activation; novel PKCs that need diacylglycerol and phospholipids; and atypical PKCs that need neither diacylglycerol nor Ca2+ (43). We found that BIS, a nonselective inhibitor of PKC isoforms, prevented Src activation in mesangial cells exposed to TGF-. However, Src activation by TGF- was unaffected by a selective inhibitor of conventional PKCs, Gö-6976 (22). These findings suggested that a novel or an atypical PKC isoform participated in the pathway by which TGF- activates Src.
Our results support the hypothesis that PKC participates in Src activation by TGF-. PKC is a novel PKC isoform with distinct mechanisms of activation and physiological functions compared with other PKC proteins (44). A role for PKC was suggested by two previous findings. First, Runyan et al. (36) found that TGF- activates PKC in HMC and that PKC is a downstream effector of TGF- signaling. Additional reports imply that PKC activates Src in other cell types and that PKC-Src constitutes a signaling motif regulating diverse phenotypic responses including collagen type I accumulation (50), organization of the actin cytoskeleton (4), and podosome assembly (46). In our present studies, a selective inhibitor of PKC, rottlerin (12), prevented TGF- from activating Src in HMC. We used rottlerin at doses that selectively block PKC (12). To further investigate the role of PKC in Src activation, we transduced mesangial cells with adenovirus expressing a dominant negative mutant of PKC (7, 8, 33). Transduction of HMC with the adenovirus expressing dominant negative PKC prevented the increase in PTyr416 Src in cells exposed to TGF-. Transduction with an adenovirus vector not expressing dominant negative PKC had no effect on TGF--induced Src activity, suggesting that a nonspecific effect of adenoviral infection was not responsible for inhibition of TGF--induced Src activity. Together, our results strongly suggest that TGF- activates Src in HMC by a novel PKC-mediated mechanism.
Src is needed for type I collagen accumulation in HMC exposed to TGF-.
A hallmark of myofibroblast transdifferentiation is accumulation of extracellular matrix, especially fibrillar collagens, leading to fibrosis. The most abundant fibrillar collagen, type I collagen, accumulates in response to many stimuli of myofibroblast transdifferentiation in vitro and in vivo (47, 48). Besides their role in fibrosis, type I collagens help control dynamic interactions between the extracellular matrix and resident cells. Some of the phenotypic responses to collagen type I accumulation include cell adhesion, chemotaxis, and migration. Because a rise in type I collagen by TGF- is a hallmark of myofibroblast transdifferentiation in HMC, we investigated whether Src participates in collagen type I induction by TGF-.
Accumulation of type I collagen is tightly regulated by transcriptional and posttranscriptional mechanisms that are highly conserved in flies, worms, and humans (18, 31). Cells synthesize and secrete all fibril-forming collagens as soluble precursors, the procollagens. The Hsp47 chaperone mediates proper folding of the procollagen molecule. Following secretion, the procollagen NH2- and COOH-terminal domains are processed by a complex array of proteases (31). Extracellular processing of the secreted type I procollagen requires at least two NH2- and COOH-terminal proteases to form a mature type I collagen trimer that self-assembles into higher order fibrils (18, 31). At this point lysyl oxidase forms covalent cross-links that stabilize the collagen-rich matrix. So accumulation of type I collagen accumulation can be roughly divided into two steps: 1) secretion of the type I procollagen molecule, and 2) posttranslational processing and association with the cell and neighboring matrix.
Our present results suggest that TGF--Src signaling regulates type I collagen secretion and the association of type I collagen with the HMC monolayer. PP2 and SU 6656 blocked both type I collagen secretion and its association with HMC. A dominant negative Src mutant also prevented the increment in collagen type I in TGF--stimulated cells. If, as we propose, TGF- activates Src by a PKC-dependent mechanism, then inhibition of PKC should block secretion and cell association of type I collagen. Indeed, our results demonstrate that rottlerin blocks secreted and cell-associated type I collagen in HMC exposed to TGF-. Dominant negative PKC also prevents collagen type I secretion and cell association in response to TGF-. Specific inhibitors of MEK1/2 that block ERK1/2 activation by TGF- prevent the increase in collagen type I secretion in mesangial cells exposed to TGF-. Similarly, Schnaper and colleagues (14, 38) documented a role for ERK1/2 in collagen type I association in TGF--treated HMC.
In conclusion, we identified a crucial role for Src in the postreceptor signals by which TGF- upregulates type I collagen, an important phenotypic response in myofibroblast transdifferentiation. Src also is required for induction of type I collagen by ET-1, another stimulus for myofibroblast transdifferentiation. Because myofibroblast transdifferentiation is driven by multiple mediators in vivo, it will be important to determine whether Src acts as a proximal postreceptor effector that integrates signals from several receptor subtypes that initiate fibrosis.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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