| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 4, 2002 as doi:10.1096/fj.02-0204fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centre for Rheumatology, Royal Free Hospital and University College School of Medicine, London NW3 2PF, UK; and
* FibroGen Inc., South San Francisco, California, USA
3Correspondence: FibroGen Inc., 225 Gateway Blvd., South San Francisco, CA 94080, USA. E-mail: aleask{at}fibrogen.com
SPECIFIC AIM
Prostacyclin and its derivatives, such as iloprost, have anti-fibrotic effects. In this report, we investigate the mechanism of action of iloprost.
PRINCIPAL FINDINGS
1. Iloprost blocks TGF-ß induced fibrosis in vivo
To investigate the potential antifibrotic effect of iloprost, we tested the ability of this prostacyclin analog to block TGF-ß-induced fibrosis in vivo. We implanted wound chambers in the back of rats and injected them every 2 days with either 1) DMSO, 2) TGF-ß, 3) iloprost, or 4) TGF-ß plus iloprost for a total of 14 days. After this period, chambers were removed and associated tissue was processed. Equal amounts of tissue were subjected to pepsin digestion, and collagen was recovered by ammonium sulfate precipitation and SDS gel electrophoresis. Wound chamber tissue was fixed, sectioned, and collagen protein was detected by staining with trichrome. Relative to chambers receiving no treatment, TGF-ß treatment of wound chambers resulted in an increase of collagen protein. TGF-ß-mediated induction of collagen protein was markedly reduced in the presence of iloprost. Addition of iloprost alone did not reduce collagen levels below basal levels observed in the DMSO-treated controls, indicating that iloprost suppressed the profibrotic effect of excess TGF-ß while leaving the normal wound healing process unaltered. We then measured the effects of iloprost on type I collagen gene expression in vivo using a transgenic mouse carrying a type I collagen-luciferase reporter gene. TGF-ß with or without iloprost was delivered by an osmotic pump implanted beneath an incisional wound. We showed that type I collagen promoter activity was greatly enhanced by addition of TGF-ß and that this effect was abolished by the addition of iloprost.
Connective tissue growth factor (CTGF) is a marker and mediator of fibrosis. We determined whether iloprost could block the TGF-ß induction of CTGF in vivo. We first subjected wound chamber tissue sections to in situ hybridization with an antisense CTGF cRNA probe. We found that whereas CTGF mRNA was not expressed in untreated tissue, addition of TGF-ß caused a marked increase in CTGF mRNA expression (Fig. 1
b). Addition of iloprost blocked the TGF-ß induction of CTGF mRNA (Fig. 1a
). Similarly, TGF-ß induced CTGF protein expression and this induction was blocked by iloprost (Fig. 1d
). Thus, iloprost suppressed the profibrotic effects of TGF-ß in vivo.
|
2. Iloprost blocks TGF-ß induction of CTGF through the protein kinase A-mediated suppression of ras/MEK/ERK
We next investigated the molecular mechanism underlying the ability of iloprost to suppress the TGF-ß-induced expression mediated by adenylate cyclase. Adenylate cyclase synthesizes cAMP, which activates protein kinase A (PKA). We found that addition of iloprost to dermal fibroblasts induced PKA activity with a dose response commensurate with the suppression of CTGF by iloprost. The PKA inhibitor (Rp)-cAMPS abolished the suppression of CTGF by iloprost. Another PKA inhibitor Rp-cAMPS that is a less potent inhibitor of PKA modestly blocked the suppression CTGF by iloprost. Thus, iloprost suppresses the induction of CTGF through PKA.
Because TGF-ß is known to induce Ras/MEK/ERK activity in other cell types, we assessed whether this pathway was required for the induction of CTGF by TGF-ß. We transfected fibroblasts with a CTGF promoter/SEAP reporter construct and cotransfected either an expression vector encoding dominant negative Ras (dnRas) or an empty expression vector as a control. dnRas suppressed the TGF-ß-mediated induction of the CTGF promoter. The MEK inhibitor U0126 blocked TGF-ß induction of the CTGF promoter. Finally, U0126 blocked the ability of TGF-ß to induce CTGF protein. Thus, ras/MEK/ERK was necessary for the TGF-ß induction of CTGF in fibroblasts.
In several systems, cAMP suppresses ras/MEK/ERK. To assess whether iloprost could be exerting its anti-fibrotic effects at least in part through PKA-dependent inhibition of ras/MEK/ERK, we serum-starved fibroblasts for 24 h, then exposed cells to TGF-ß for varying lengths of time. Cell extracts were subjected to Western blot with anti-ERK1 and 2, and anti-phosphorylated ERK1 and 2. ERK1 and 2 were constitutively phosphorylated in dermal fibroblasts and there was a two- to threefold increase in ERK phosphorylation after a 15 min incubation with TGF-ß (Fig. 2
). We then exposed cells to TGF-ß (10 ng/mL) with or without iloprost (0.5 ng/mL) for 15 min. iloprost suppressed basal and induced ERK phosphorylation (Fig. 2)
. We performed experiments in parallel to show that treatment with iloprost blocked the induction of CTGF by TGF-ß (not shown). We added various amounts of the PKA inhibitor Rp-8Br cAMPS 2 h before addition of TGF-ß and iloprost (Fig. 2)
. We found that pretreatment with Rp-8Br cAMPS blocked the ability of iloprost to suppress ERK phosphorylation. Thus, given that ras/MEK/ERK was required for the TGF-ß induction of CTGF and that iloprost suppressed activity of this MAPK cascade, we concluded that iloprost suppresses TGF-ß-induced fibrosis at least in part through the PKA-dependent inhibition of ras/MEK/ERK.
|
3. Ras/MEK/ERK pathway does not directly affect SMAD signaling in fibroblasts
Induction of CTGF by TGF-ß requires SMAD 3/4 and a SMAD binding element within the CTGF promoter. SMAD 2 is not involved in this process. Endogenous SMAD3 needs to be phosphorylated by the TGF-ß receptor in a ligand-dependent fashion for it to be capable of activating transcription. To assess whether activation of the Ras/MEK/ERK pathway was directly affecting SMAD3 signaling in fibroblasts, we serum-starved fibroblasts for 24 h, then incubated cells in the presence or absence of U0126 for 45 min before adding TGF-ß for 15 min. Cell layers were lysed and subjected to Western blot analysis with an anti-phospho-SMAD2/3 antibody. TGF-ß induced SMAD phosphorylation, which was not reduced by U0126.
Transfection of SMAD3/4 into cells allows the activation of target genes, including CTGF, in a ligand-independent fashion. U0126 or transfection of dnRAS did not block the ability of SMAD3 and 4 to trans-activate the CTGF promoter in NIH 3T3 fibroblasts. We concluded that although ras/MEK/ERK seems to be required for the SMAD-dependent activation of the CTGF promoter, ras/MEK/ERK seems not to directly affect the ability of SMADs to be activated by phosphorylation or to activate promoter activity.
DISCUSSION
Fibrosis is a general term applied to a large group of diseases characterized by excessive matrix deposition leading to organ dysregulation and failure. There are currently no effective anti-fibrotic therapies. TGF-ß is a profibrotic cytokine, as it induces myofibroblasts and matrix synthesis. Furthermore, inhibition of TGF-ß reduces drug-induced fibrosis in animal models. However, given TGF-ß’s widespread role in physiology, more specific targets for anti-fibrotic intervention might be beneficial. CTGF normally is not expressed in fibroblasts unless cells are exposed to TGF-ß, but is constitutively overexpressed in fibrosis. Although intradermal injection of TGF-ß in mice causes transient fibrosis and injection of CTGF has no effect, coinjection of TGF-ß and CTGF cause sustained fibrosis. CTGF is thus both a marker and a mediator of the fibrotic phenotype. Blocking CTGF expression might provide a selective approach to developing anti-fibrotic drugs.
Iloprost is a synthetic, stable prostacyclin derivative widely used to treat Raynaud’s phenomenon in scleroderma patients. We recently showed that iloprost suppresses TGF-ß induction of collagen and CTGF and that this process is dependent on adenylate cyclase. In this report, we show that iloprost blocks the fibrotic effect of TGF-ß in vivo.
Outside the setting of fibrosis, TGF-ß has been shown to activate a series of signaling cascades such as Ras/MEK/ERK. In this report, we have showed that 1) Ras and MEK are required for the induction of CTGF by TGF-ß, 2) TGF-ß is able to enhance phosphorylation of ERK, and 3) iloprost suppressed the induction of ERK phosphorylation by TGF-ß in a PKA-dependent fashion. Collectively, these results suggest that in fibroblasts ras/MEK/ERK promotes, whereas PKA antagonizes, TGF-ß-dependent fibrosis (Fig. 3
).
|
The TGF-ß induction of CTGF and collagen requires SMAD3; mutating a functional SMAD binding sites completely abolished the induction CTGF by TGF-ß. Thus, Ras/MEK/ERK is not able to induce CTGF expression in the absence of SMAD signaling. SMADs are not thought to activate transcription themselves, but rather are thought to act by promoting the formation of active transcriptional complexes. Therefore, ras/MEK/ERK might modulate transcription factors that act with SMADs to activate gene expression. We recently identified a motif other than the SMAD recognition sequence that is necessary for the TGF-ß induction of CTGF. Preliminary experiments suggest that ras/MEK/ERK could be acting through this additional promoter element. Inhibiting ras/MEK/ERK or identifying how TGF-ß stimulates this pathways may provide a more selective way of blocking the profibrotic effects of TGF-ß but leave other effects of TGF-ß intact.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0204fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0204fje 
This article has been cited by other articles:
|
|
 |
|
  V. Haydont, B. L. Riser, J. Aigueperse, and M.-C. Vozenin-Brotons Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-{beta}1 Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1332 - C1341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kamio, T. Sato, X. Liu, H. Sugiura, S. Togo, T. Kobayashi, S. Kawasaki, X. Wang, L. Mao, Y. Ahn, et al. Prostacyclin analogs stimulate VEGF production from human lung fibroblasts in culture Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1226 - L1232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Huang, S. H. Wettlaufer, C. M. Hogaboam, K. R. Flaherty, F. J. Martinez, J. L. Myers, T. V. Colby, W. D. Travis, G. B. Toews, and M. Peters-Golden Variable Prostaglandin E2 Resistance in Fibroblasts from Patients with Usual Interstitial Pneumonia Am. J. Respir. Crit. Care Med., January 1, 2008; 177(1): 66 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Haydont, C. Bourgier, M. Pocard, A. Lusinchi, J. Aigueperse, D. Mathe, J. Bourhis, and M.-C. Vozenin-Brotons Pravastatin Inhibits the Rho/CCN2/Extracellular Matrix Cascade in Human Fibrosis Explants and Improves Radiation-Induced Intestinal Fibrosis in Rats Clin. Cancer Res., September 15, 2007; 13(18): 5331 - 5340. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Liu, X. Shi-wen, L. Kennedy, D. Pala, Y. Chen, M. Eastwood, D. E. Carter, C. M. Black, D. J. Abraham, and A. Leask FAK Is Required for TGFbeta-induced JNK Phosphorylation in Fibroblasts: Implications for Acquisition of a Matrix-remodeling Phenotype Mol. Biol. Cell, June 1, 2007; 18(6): 2169 - 2178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Leask TGF{beta}, cardiac fibroblasts, and the fibrotic response Cardiovasc Res, May 1, 2007; 74(2): 207 - 212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Leask and D. J. Abraham All in the CCN family: essential matricellular signaling modulators emerge from the bunker J. Cell Sci., December 1, 2006; 119(23): 4803 - 4810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Liu, S. Q. Sun, A. Hassid, and R. S. Ostrom cAMP Inhibits Transforming Growth Factor-beta-Stimulated Collagen Synthesis via Inhibition of Extracellular Signal-Regulated Kinase 1/2 and Smad Signaling in Cardiac Fibroblasts Mol. Pharmacol., December 1, 2006; 70(6): 1992 - 2003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Lovgren, L. A. Jania, J. M. Hartney, K. K. Parsons, L. P. Audoly, G. A. FitzGerald, S. L. Tilley, and B. H. Koller COX-2-derived prostacyclin protects against bleomycin-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L144 - L156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Shi-Wen, F. Rodriguez-Pascual, S. Lamas, A. Holmes, S. Howat, J. D. Pearson, M. R. Dashwood, R. M. du Bois, C. P. Denton, C. M. Black, et al. Constitutive ALK5-Independent c-Jun N-Terminal Kinase Activation Contributes to Endothelin-1 Overexpression in Pulmonary Fibrosis: Evidence of an Autocrine Endothelin Loop Operating through the Endothelin A and B Receptors. Mol. Cell. Biol., July 1, 2006; 26(14): 5518 - 5527. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Shi-wen, L. A. Stanton, L. Kennedy, D. Pala, Y. Chen, S. L. Howat, E. A. Renzoni, D. E. Carter, G. Bou-Gharios, R. J. Stratton, et al. CCN2 Is Necessary for Adhesive Responses to Transforming Growth Factor-beta1 in Embryonic Fibroblasts J. Biol. Chem., April 21, 2006; 281(16): 10715 - 10726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. R. A. Johnson, J. K. Burgess, Q. Ge, M. Poniris, S. Boustany, S. M. Twigg, and J. L. Black Connective Tissue Growth Factor Induces Extracellular Matrix in Asthmatic Airway Smooth Muscle Am. J. Respir. Crit. Care Med., January 1, 2006; 173(1): 32 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Murakami, N. Nagaya, T. Itoh, M. Kataoka, T. Iwase, T. Horio, Y. Miyahara, Y. Sakai, K. Kangawa, and H. Kimura Prostacyclin agonist with thromboxane synthase inhibitory activity (ONO-1301) attenuates bleomycin-induced pulmonary fibrosis in mice Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L59 - L65. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, X. Shi-wen, J. van Beek, L. Kennedy, M. McLeod, E. A. Renzoni, G. Bou-Gharios, S. Wilcox-Adelman, P. F. Goetinck, M. Eastwood, et al. Matrix Contraction by Dermal Fibroblasts Requires Transforming Growth Factor-{beta}/Activin-Linked Kinase 5, Heparan Sulfate-Containing Proteoglycans, and MEK/ERK: Insights into Pathological Scarring in Chronic Fibrotic Disease Am. J. Pathol., December 1, 2005; 167(6): 1699 - 1711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Nasrallah and R. L. Hebert Prostacyclin signaling in the kidney: implications for health and disease Am J Physiol Renal Physiol, August 1, 2005; 289(2): F235 - F246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, D. J. Abraham, X. Shi-wen, J. D. Pearson, C. M. Black, K. M. Lyons, and A. Leask CCN2 (Connective Tissue Growth Factor) Promotes Fibroblast Adhesion to Fibronectin Mol. Biol. Cell, December 1, 2004; 15(12): 5635 - 5646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. UNNIKRISHNAN and B. A. BURLEIGH Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi-mediated response FASEB J, November 1, 2004; 18(14): 1625 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Shi-Wen, Y. Chen, C. P. Denton, M. Eastwood, E. A. Renzoni, G. Bou-Gharios, J. D. Pearson, M. Dashwood, R. M. du Bois, C. M. Black, et al. Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts Mol. Biol. Cell, June 1, 2004; 15(6): 2707 - 2719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Shi-wen, S. L. Howat, E. A. Renzoni, A. Holmes, J. D. Pearson, M. R. Dashwood, G. Bou-Gharios, C. P. Denton, R. M. du Bois, C. M. Black, et al. Endothelin-1 Induces Expression of Matrix-associated Genes in Lung Fibroblasts through MEK/ERK J. Biol. Chem., May 28, 2004; 279(22): 23098 - 23103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. LEASK and D. J. ABRAHAM TGF-{beta} signaling and the fibrotic response FASEB J, May 1, 2004; 18(7): 816 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Holmes, D. J. Abraham, Y. Chen, C. Denton, X. Shi-wen, C. M. Black, and A. Leask Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1 J. Biol. Chem., October 24, 2003; 278(43): 41728 - 41733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Iwanciw, M. Rehm, M. Porst, and M. Goppelt-Struebe Induction of Connective Tissue Growth Factor by Angiotensin II: Integration of Signaling Pathways Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1782 - 1787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Ivarsen, T Laurberg, and T Moller-Pedersen Characterisation of corneal fibrotic wound repair at the LASIK flap margin Br. J. Ophthalmol., October 1, 2003; 87(10): 1272 - 1278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Leask, A. Holmes, C. M. Black, and D. J. Abraham Connective Tissue Growth Factor Gene Regulation. REQUIREMENTS FOR ITS INDUCTION BY TRANSFORMING GROWTH FACTOR-beta 2 IN FIBROBLASTS J. Biol. Chem., April 4, 2003; 278(15): 13008 - 13015. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
