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Induction of Connective Tissue Growth Factor by Activation of Heptahelical Receptors

2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês

10.1074/jbc.m000976200

ISSN

1083-351X

Autores

Angelika F. Hahn, Juliane Heusinger‐Ribeiro, T. Lanz, Susanne Zenkel, Margarete Goppelt‐Struebe,

Tópico(s)

Bone health and treatments

Resumo

Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction ofCTGF by transforming growth factor beta (TGF-β) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediatedCTGF induction was due to activation of 5-HT2Areceptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-β. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction ofCTGF by LPA and TGF-β. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulatedCTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGFexpression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants ofCTGF expression induced by LPA and 5-HT, and also by TGF-β. Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction ofCTGF by transforming growth factor beta (TGF-β) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediatedCTGF induction was due to activation of 5-HT2Areceptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-β. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction ofCTGF by LPA and TGF-β. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulatedCTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGFexpression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants ofCTGF expression induced by LPA and 5-HT, and also by TGF-β. connective tissue growth factor cyclooxygenase lysophosphatidic acid mitogen-activated protein kinase serotonin transforming growth factor β pertussis toxin fetal calf serum 4-morpholinepropanesulfonic acid glyceraldehyde-3-phosphate dehydrogenase Connective tissue growth factor (CTGF)1 belongs to the family of low affinity insulin-like growth factor binding proteins, which consists of Mac25, the nov oncogenes, and cyr61 (1Kim H.S. Nagalla S.R. Oh Y. Wilson E. Roberts Jr., C.T. Rosenfeld R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12981-12986Crossref PubMed Scopus (284) Google Scholar), and is also classified as a member of the CCN (CYR61, CTGF, and NOV) family (2Bork P. FEBS Lett. 1993; 327: 125-130Crossref PubMed Scopus (552) Google Scholar, 3Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar). These proteins share structural homologies and function as growth modulators. CTGF was first purified from conditioned medium of human umbilical vein endothelial cells and shown to account for much of the bioactivity previously attributed to platelet-derived growth factor (4Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. J. Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (811) Google Scholar). Recent data obtained with aortic smooth muscle cells and breast cancer cells demonstrated that CTGF may act as a mediator of growth arrest and apoptosis (5Murphy M. Godson C. Cannon S. Kato S. Mackenzie H.S. Martin F. Brady H.R. J. Biol. Chem. 1999; 274: 5830-5834Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 6Hishikawa K. Oemar B.S. Tanner F.C. Nakaki T. Fujii T. Luscher T.F. Circulation. 1999; 100: 2108-2112Crossref PubMed Scopus (93) Google Scholar, 7Hishikawa K. Oemar B.S. Tanner A.M. Nakaki T. Luscher T.F. Fujii T. J. Biol. Chem. 1999; 274: 37461-37466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In fibroblasts, it is most potently induced by transforming growth factor beta (TGF-β) (8Grotendorst G.R. Okochi H. Hayashi N. Cell Growth Differ. 1996; 7: 469-480PubMed Google Scholar). It stimulates fibroblast cell proliferation and mediates TGF-β-induced anchorage-independent growth (9Kothapalli D. Frazier K.S. Welply A. Segarini P.R. Grotendorst G.R. Cell Growth Differ. 1997; 8: 61-68PubMed Google Scholar). Furthermore, CTGF is a potent stimulator of extracellular matrix synthesis (10Frazier K. Williams S. Kothapalli D. Klapper H. Grotendorst G.R. J. Investig. Dermatol. 1996; 107: 404-411Abstract Full Text PDF PubMed Scopus (673) Google Scholar, 11Duncan M.R. Frazier K.S. Abramson S. Williams S. Klapper H. Huang X. Grotendorst G.R. FASEB J. 1999; 13: 1774-1786Crossref PubMed Scopus (579) Google Scholar). Elevated levels of CTGF are found in fibrotic lesions (e.g.Refs. 12Igarashi A. Nashiro K. Kikuchi K. Sato S. Ihn H. Fujimoto M. Grotendorst G.R. Takehara K. J. Investig. Dermatol. 1996; 106: 729-733Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 13Oemar B.S. Werner A. Garnier J.M. Do D.D. Godoy N. Nauck M. Marz W. Rupp J. Pech M. Luscher T.F. Circulation. 1997; 95: 831-839Crossref PubMed Scopus (290) Google Scholar, 14Dammeier J. Brauchle M. Falk W. Grotendorst G.R. Werner S. Int. J. Biochem. Cell Biol. 1998; 30: 909-922Crossref PubMed Scopus (146) Google Scholar) and suggested to be functionally involved in the development and progression of fibrotic diseases. In the kidney,CTGF mRNA levels were elevated in the majority of biopsies obtained from patients with various types of renal diseases characterized by glomerulosclerosis and tubulointerstitial fibrosis (15Ito Y. Aten J. Bende R.J. Oemar B.S. Rabelink T.J. Weening J.J. Goldschmeding R. Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). In the glomerulus, basal expression of CTGF was detected in epithelial podocytes. In the inflamed glomerulus, CTGF was up-regulated in proliferating epithelial cells and also observed in mesangial cells. Mesangial cells cultured in vitro express basal levels ofCTGF mRNA, which are further increased by TGF-β (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar). In accordance with elevated CTGF expression in diabetic glomerulosclerosis (15Ito Y. Aten J. Bende R.J. Oemar B.S. Rabelink T.J. Weening J.J. Goldschmeding R. Kidney Int. 1998; 53: 853-861Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar), elevation of glucose levels enhancedCTGF mRNA levels in cultured mesangial cells (5Murphy M. Godson C. Cannon S. Kato S. Mackenzie H.S. Martin F. Brady H.R. J. Biol. Chem. 1999; 274: 5830-5834Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Up-regulation of CTGF by glucose was blocked by anti-TGF-β antibodies, confirming CTGF as a downstream target of TGF-β in mesangial cells (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar). Besides TGF-β, CTGF itself was able to induce its own mRNA expression (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar). Mesangial cells are thus target cells of CTGF, as also shown by the induction of extracellular matrix proteins (fibronectin and collagen I and IV) (5Murphy M. Godson C. Cannon S. Kato S. Mackenzie H.S. Martin F. Brady H.R. J. Biol. Chem. 1999; 274: 5830-5834Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). CTGF belongs to the group of proteins coded for by immediate early response genes, which in general are induced by a variety of different mediators. As an example, cyclooxygenase-2 (Cox-2) is induced by growth factors, cytokines, and low molecular mediators acting via serpentine receptors (17Goppelt-Struebe M. Prostaglandins Leukot. Essent. Fatty Acids. 1995; 52: 213-222Abstract Full Text PDF PubMed Scopus (139) Google Scholar). This prompted us to further investigate the regulation of CTGFmRNA and protein expression in renal mesangial cells. Lysophosphatidic acid (LPA) is generated by cleavage of glycerophospholipids in membranes of stimulated cells. Increased release of LPA is observed in tissue injury, inflammation, and neoplasia (18Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar). Activated platelets are an abundant source of LPA, and high levels of the lysophospholipid (2–20 μm) are detectable in serum (19Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar). Via binding to seven transmembrane receptors (edg receptors), LPA modulates cell proliferation and differentiation and mediates cellular effects such as chemotaxis, adhesion, contraction, or aggregation, which are related to cytoskeletal rearrangements (18Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar). Treatment of mesangial cells with LPA led to contraction of the cells (20Inoue C.N. Forster H.G. Epstein M. Circ. Res. 1995; 77: 888-896Crossref PubMed Scopus (46) Google Scholar) and stimulated proliferation (21Gaits F. Salles J.-P. Chap H. Kidney Int. 1997; 51: 1022-1027Abstract Full Text PDF PubMed Scopus (34) Google Scholar,22Inoue C.N. Ko Y.H. Guggino W.B. Forster H.G. Epstein M. Proc. Soc. Exp. Biol. Med. 1997; 216: 370-379Crossref PubMed Scopus (34) Google Scholar). Proliferation was shown to be mediated by the induction of the expression of the immediate early gene Egr-1 (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar). Likewise,Cox-2, another example of an early response gene, was rapidly induced by LPA in mesangial cells (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar). LPA-mediated induction of the early response genes was pertussis toxin-sensitive,i.e. mediated by G proteins of the Gi type. Furthermore, activation of heptahelical receptors coupling to pertussis toxin-insensitive G proteins also led to the induction of these early response genes as exemplified by serotonin (5-HT) (24Stroebel M. Goppelt-Struebe M. J. Biol. Chem. 1994; 269: 22952-22957Abstract Full Text PDF PubMed Google Scholar, 25Goppelt-Struebe M. Stroebel M. J. Cell. Physiol. 1998; 175: 341-347Crossref PubMed Scopus (34) Google Scholar). Activation of p42/44 MAP kinase was a common signaling module in both pathways: the kinase was rapidly activated by LPA or 5-HT, and inhibition of p42/44 MAP kinase prevented induction of Cox-2 orEgr-1 (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar, 26Goppelt-Struebe M. Hahn A. Stroebel M. Reiser C.O. Biochem. J. 1999; 339: 329-334Crossref PubMed Scopus (28) Google Scholar). It was thus tempting to speculate that the early response gene CTGF might be another target of LPA and/or 5-HT in mesangial cells. Based on the previous studies on the induction of Egr-1 and Cox-2, rat mesangial cells were used to investigate the induction of CTGF by activation of heptahelical receptors and to delineate the signaling pathways responsible for CTGF induction, which have not yet been described in detail in any cell type. Recombinant human TGF-β was obtained from TEBU, Frankfurt, Germany. PD-98059 was from Calbiochem, Bad Soden, Germany. LPA, serotonin (5-HT), and cytochalasin D were from Sigma, Deisenhofen, Germany. Pertussis toxin (PTX) was from Biomol, Hamburg, Germany. Cell culture reagents were from Biochrom, Berlin, Germany. FCS was from Life Technologies, Inc., Eggenstein, Germany. Toxin B from Clostridium difficile was kindly provided by Drs. F. Hofmann and K. Aktories, Freiburg, Germany. Y-27632 was kindly provided by Yoshitomi Pharmaceutical Industries, Osaka, Japan. Rat mesangial cells were isolated as described (27Lovett D.H. Ryan J.L. Sterzel R.B. J. Immunol. 1983; 131: 2830-2836PubMed Google Scholar) and were grown in Dulbecco's modified Eagle's medium supplemented with 2 mml-glutamine, 5 μg/ml insulin, 4.5 g/liter glucose, 100 units/ml penicillin, and 100 μg/ml streptomycin containing 10% FCS. Mesangial cells (0.5–1.0 × 106 cells/10 ml) were plated in 100-mm Petri dishes in medium with 10% FCS. At subconfluency (after 3–4 days) cells were serum-starved in Dulbecco's modified Eagle's medium containing 0.5% FCS for 2–3 days. Northern blot analysis was performed as described previously (24Stroebel M. Goppelt-Struebe M. J. Biol. Chem. 1994; 269: 22952-22957Abstract Full Text PDF PubMed Google Scholar). After stimulation for the indicated times, total RNA was extracted according to the protocol of Chomczynski and Sacchi (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar) with minor alterations. Usually, RNA yield was about 30–40 μg/10-cm Petri dish. Separation of total RNA (10 μg/lane) was achieved by use of 1.2% agarose gels containing 1.9% formaldehyde with 1 × MOPS as gel running buffer. Separated RNA was transferred to nylon membranes by capillary blotting and fixed by baking at 80 °C for 2 h. Hybridization was performed with cDNA probes labeled with [32P]dCTP using the NonaPrimer kit from Appligene, Heidlberg, Germany. The specific Cox-2 probe was a 1.156-kilobase EcoRI fragment from the 5′-end of mouse cDNA (29DeWitt D.L. Meade E.A. Arch. Biochem. Biophys. 1993; 306: 94-102Crossref PubMed Scopus (230) Google Scholar). A cDNA specific for CTGF (full-length cDNA of human CTGF) was kindly provided by N. Wahab, London, UK (30Mason R. Li X.J. Wahab N.A. J. Am. Soc. Nephrol. 1997; 8: 642AGoogle Scholar). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe was obtained with a 500-base pair reverse-transcribed fragment. DNA/RNA hybrids were detected by autoradiography using Kodak X-Omat AR film. As a control for equal loading of the gels, the housekeeping gene GAPDH or the 18 S rRNA were hybridized or the blotted 18 S rRNA was stained with methylene blue (0.04% in 500 mm sodium acetate, pH 5.2) and directly quantitated by densitometry. Quantitative analysis of the autoradiographs was performed by densitometric scanning (Froebel, Wasserburg, Germany). All values were corrected for differences in RNA loading by calculating the ratio of the specific bands toGAPDH or 18 S rRNA expression. The two-sided Student'st test for paired samples was used to calculate significant differences. Cellular proteins were isolated using radioimmune precipitation buffer (50 mm Tris/HCl, pH 7.5, 1% (v/v) Triton X-100, 0.1% (w/v) deoxycholic acid, 0.1% (w/v) SDS, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium vanadate, 14 μg/ml aprotinin). For Western blot analysis, 75 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide), transferred onto a polyvinylidene difluoride membrane (Pall Biosupport Division, Dreieich, Germany) and probed with an antibody directed against mouse CTGF. The antibody was kindly provided by S. Werner, Zurich, Switzerland (31Dammeier J. Beer H.D. Brauchle M. Werner S. J. Biol. Chem. 1998; 273: 18185-18190Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Cells were cultured and growth-arrested on glass 8-well multitest slides (ICN, Cleveland, OH) placed in a Petri dish. After stimulation, the cells were fixed with 3% paraformaldehyde in phosphate-buffered saline for 10 min and then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 7 min at room temperature. After washing, the actin cytoskeleton was stained with rhodamine-phalloidin complex (Molecular Probes, Leiden, The Netherlands) for 20 min. TGF-β has been characterized as a potent inducer of CTGF in different cell types, among them human mesangial cells (5Murphy M. Godson C. Cannon S. Kato S. Mackenzie H.S. Martin F. Brady H.R. J. Biol. Chem. 1999; 274: 5830-5834Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). This was confirmed when rat mesangial cells were treated with TGF-β with subsequent analysis of CTGFmRNA expression (Fig. 1 A). A 2-fold stimulation was observed after 2 h (1.8 ± 0.4,n = 3, means ± S.D.). CTGF mRNA levels were further increased at later time points (4 h, 3.7 ± 0.8, n = 4, means ± S.D., p < 0.005). Two activators of heptahelical receptors, serotonin (5-HT) and lysophosphatidic acid (LPA) showed a similar but more transient response. Maximal stimulation of CTGF by LPA was observed after 1 h (Fig. 1 B). Stimulation was 2.0 ± 0.4-fold (means ± S.D., n = 9, p< 0.05) with 10 μm LPA. A similar stimulation was observed with 5-HT (1.9 ± 0.7, n = 7, means ± S.D., p < 0.05, stimulation time 2 h). Induction of CTGF by LPA was concentration-dependent. Micromolar concentrations were necessary and sufficient to induce CTGF mRNA induction (Fig. 1 C). Increased proliferation of mesangial cells and the induction of the early response genes Egr-1 andCox-2 were observed in the same concentration range of LPA (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar). LPA concentrations were within the range reported to occur in serum (2–20 μm (19Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar)). Higher concentrations were not used to avoid nonspecific effects of the lysophospholipid. When mesangial cells were incubated with LPA plus TGF-β, the increase inCTGF mRNA expression was additive (Fig. 1 D). Low concentrations of LPA, which by themselves did not induceCTGF expression, did not significantly augment TGF-β-mediated induction of CTGF. Additivity was also observed with TGF-β and 5-HT (data not shown). Treatment of mesangial cells with the inhibitor of protein synthesis cycloheximide revealed the dynamics of basal CTGF mRNA expression: mRNA levels were increased with time most likely due to the inhibition of the synthesis of degrading enzymes. In line with the characterization of CTGF as an immediate early response gene (e.g.Ref. 8Grotendorst G.R. Okochi H. Hayashi N. Cell Growth Differ. 1996; 7: 469-480PubMed Google Scholar), LPA-mediated induction of CTGF was not inhibited in the presence of cycloheximide but strongly increased (Fig.1 E). CTGF is a secreted protein, and it was thus attempted to detect CTGF protein in cell culture supernatants. No CTGF protein was detectable even if the cells were stimulated with LPA or TGF-β for up to 20 h and the cell culture supernatants were concentrated more than 30-fold. Analysis of cellular homogenates by Western blot analysis with a specific antibody directed against mouse CTGF (31Dammeier J. Beer H.D. Brauchle M. Werner S. J. Biol. Chem. 1998; 273: 18185-18190Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) revealed the time-dependent induction of a protein of about 38 kDa when the cells were treated with LPA or with TGF-β (Fig.2 A). This suggested that CTGF was either retained within the cells, or more likely remained attached to the cells as has been shown for human mesangial cells (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar). A nonspecific band was detected at about 90 kDa, which was not regulated. This band was used as reference for the densitometric quantification of the Western blots (Fig. 2 B). The time course of protein expression corresponded to the time course of mRNA induction. Co-incubation of mesangial cells with LPA and TGF-β resulted in an increased expression of CTGF protein that was additive rather than synergistic (Fig. 2 C). A second nonspecific band of a protein of about 33 kDa was detected on most blots, but with varying intensity (see also 5D). As described before (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar) expression of LPA-mediatedCox-2 mRNA was strongly reduced by pretreatment of the cells with pertussis toxin (PTX), whereas 5-HT-mediated induction of this early response gene was not affected by PTX, indicating coupling to different types of G proteins (Fig.3 A). Preincubation with PTX for 18 h did not significantly affect CTGF induction by LPA or 5-HT, indicating predominant activation of G proteins of the Gq or G12/13 family in both signaling pathways (Fig. 3, A and B). In line with these results, 5-HT-mediated induction of CTGF was prevented by preincubation with ketanserin, a specific inhibitor of 5-HT2A receptors, which couple to Gq proteins (Fig. 3 C). Co-incubation of mesangial cells with 5-HT and LPA did not further enhance CTGF expression, whereasCox-2 expression was increased (Fig. 3 A and Ref.23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar). LPA and 5-HT, both activate p42/44 mitogen-activated protein (MAP) kinases in mesangial cells (26Goppelt-Struebe M. Hahn A. Stroebel M. Reiser C.O. Biochem. J. 1999; 339: 329-334Crossref PubMed Scopus (28) Google Scholar, 32Goppelt-Struebe M. Fickel S. Reiser C.O.A. Biochem. J. 2000; 349(Prt 1): 217-224Crossref Google Scholar). These kinases were shown to be essential parts of LPA- and 5-HT-mediated induction of the early response genes Egr-1 and Cox-2 in mesangial cells (Fig. 4 A and Refs. 23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar and 25Goppelt-Struebe M. Stroebel M. J. Cell. Physiol. 1998; 175: 341-347Crossref PubMed Scopus (34) Google Scholar). Treatment of mesangial cells with the MEK inhibitor PD-98059 led to a concentrationdependent inhibition of MAP kinase activity reaching over 90% inhibition at a concentration of 20 μm(32Goppelt-Struebe M. Fickel S. Reiser C.O.A. Biochem. J. 2000; 349(Prt 1): 217-224Crossref Google Scholar). The basal expression of CTGF was reduced by about 10% when the cells were incubated with 10 or 20 μm PD-98059 (Fig. 4, A and B). Induction of CTGFby LPA or 5-HT, however, was not impaired by PD-98059 and, thus, was independent of p42/44 MAP kinase activation. Rho proteins have been characterized as downstream mediators of LPA signaling in many cellular systems (e.g. Refs. 33Mackay D.J.G. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar and 34Seasholtz T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (206) Google Scholar). Treatment of mesangial cells for 3 h with various concentrations of toxin B, an inhibitor of RhoA, Rac1, and Cdc42, led to a concentration-dependent inhibition of both basal and LPA-induced CTGF expression (Fig. 5 A). Reduction ofCTGF levels by 10 ng/ml toxin B was complete, and no expression was detectable even at longer exposure times of the blot membranes (not shown). Likewise, 5-HT-mediated induction ofCTGF was sensitive to treatment with toxin B (Fig.5 B). Induction of the early response gene Cox-2was also reduced but to a lesser extent (Fig. 5, A andB). Involvement of RhoA in LPA-mediated signaling was shown by the inhibitor Y-27632, which specifically interferes with Rho kinase, a downstream target of RhoA (35Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Meakawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Crossref PubMed Scopus (411) Google Scholar). The inhibitor reduced LPA-mediated induction of CTGF by about 50% (51.8 ± 16.0, means ± S.D., n = 3, p < 0.05; Fig. 5 C). It also interfered with TGF-β signaling, as did toxin B (see below). Inhibition of CTGF expression by toxin B was also observed at the protein level (Fig. 5 D). Inhibition of Rho family proteins strongly affected the actin cytoskeleton. Mesangial cells in culture show a high degree of actin filaments organized in stress fibers (Fig. 6). Treatment of mesangial cells with toxin B led to time-dependent changes in morphology due to a disorganization of the actin cytoskeleton. A more rapid destruction of the stress fibers was observed when the cells were treated with cytochalasin D. Cytoskeletal rearrangement as a possible explanation for the effect of toxin B on CTGF mRNA expression was investigated by comparison of toxin B and cytochalasin D. Pretreatment of mesangial cells for 1 h with toxin B (5 ng/ml) completely inhibited basal and TGF-β- or LPA-mediated induction ofCTGF, whereas treatment with cytochalasin (1 μg/ml) partially reduced basal and stimulated CTGF induction (Fig.7). The effect of cytochalasin D was concentration-dependent, 0.5 μg/ml being less effective than 1 μg/ml, with no further inhibition of CTGFexpression when the concentration of cytochalasin D was increased from 1 to 2 μg/ml (data not shown).Figure 7Interference of cytochalasin D with CTGF mRNA expression. A, mesangial cells were preincubated with toxin B (5 ng/ml, ToxB) or cytochalasin D (1 μg/ml, CytoD) for 1 h and then further incubated with LPA (10 μm) for 1 h or TGF-β for 4 h. B, to compare the CTGFexpression in different experiments, expression of CTGFafter stimulation with LPA after 1 h or TGF-β after 4 h was set to 100%. Data are means ± S.D. of four (LPA) and three (TGF-β) experiments. Inhibition was significant withp < 0.05 (*); Student's t test for paired samples was used.View Large Image Figure ViewerDownload (PPT) The low molecular weight mediators LPA and 5-HT regulate mesangial cell contraction, proliferation, and gene induction and thus play a role in the control of glomerular hemodynamics and the progression of glomerular nephritis (36Verbeke M. Smollich B. VanDeVoorde J. DeRidder L. Lameire N. J. Am. Soc. Nephrol. 1996; 7: 621-627PubMed Google Scholar, 37Inoue C.N. Epstein M. Forster H.G. Hotta O. Kondo Y. Iinuma K. Clin. Sci. 1999; 96: 431-436Crossref PubMed Google Scholar). Furthermore, 5-HT has been related to increased matrix production in mesangial cells by induction of TGF-β and enhanced synthesis of type IV collagen (38Kasho M. Sakai M. Sasahara T. Anami Y. Matsumara T. Takemura T. Matsuda H. Kobori S. Shichiri M. Kidney Int. 1998; 54: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Induction ofCTGF by LPA and 5-HT in mesangial cells further relates these mediators to the development and progression of renal fibrosis. In mesangial cells, CTGF has primarily been characterized as a downstream mediator of TGF-β, but was also induced in an autocrine manner by recombinant CTGF (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar). The data presented characterize activation of pertussis toxin-insensitive heptahelical receptors by LPA and 5-HT as novel signaling pathway to mediate CTGFinduction. Induction was transient with maximal mRNA levels reached after 1 to 2 h. Similar kinetics were observed recently, when fibroblasts were stimulated by factor VIIa and thrombin (39Pendurthi U.R. Allen K.E. Ezban M. Rao V.M. J. Biol. Chem. 2000; 275: 14632-14641Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), whereas des-Arg10-kallidin augmented CTGF mRNA levels more slowly, due to message stabilization (40Ricupero D.A. Romero J.R. Rishikof D.C. Goldstein R.H. J. Biol. Chem. 2000; 275: 12475-12480Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Activation of heptahelical receptors may thus differentially affect CTGFexpression, possibly dependent on the cell type or the coupling to different downstream signaling pathways. CTGF induction by LPA or 5-HT was insensitive to pertussis toxin, suggesting involvement of G proteins of the Gq/11 or G12/13 family. Regarding pertussis toxin-insensitive G proteins, LPA receptors seem to couple primarily to G12/13 proteins (e.g. Refs.41Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar or 42Kranenburg O. Poland M. van Horck F.P. Drechsel D. Hall A. Moolenaar W.H. Mol. Biol. Cell. 1999; 10: 1851-1857Crossref PubMed Scopus (273) Google Scholar and citations therein), suggesting that this type of G protein might also be involved in LPA-mediated induction of CTGF. 5-HT2A receptors have been characterized on mesangial cells to mediate the mitogenic effects of 5-HT as well as induction of immediate early response genes (25Goppelt-Struebe M. Stroebel M. J. Cell. Physiol. 1998; 175: 341-347Crossref PubMed Scopus (34) Google Scholar, 43Nebigil C.G. Garnovskaya M.N. Spurney R.F. Raymond J.R. Am. J. Physiol. 1995; 268: F122-F127PubMed Google Scholar, 44Goppelt-Struebe M. Stroebel M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 240-247Crossref PubMed Scopus (16) Google Scholar). Consistently, these effects were pertussis toxin-insensitive in line with coupling of 5-HT2A receptors to Gq/11 proteins. In contrast to the induction of CTGF, LPA-mediated induction of the transient expression of early response genes Egr-1 orCox-2 was pertussis toxin-sensitive in mesangial cells (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar) as was c-fos induction in fibroblasts (45Cook S.J. Aziz N. McMahon M. Mol. Cell. Biol. 1999; 19: 330-341Crossref PubMed Scopus (164) Google Scholar), indicating involvement of G proteins of the Gi type. LPA receptors have not yet been characterized in mesangial cells, and it is thus not clear whether pertussis toxin-sensitive and -insensitive effects are mediated by different receptors or by differential coupling of G proteins. Previous studies have shown activation of p42/44 MAP kinases in mesangial cells within 2 min by LPA, which was sensitive to pertussis toxin, whereas activation by 5-HT was pertussis toxin-insensitive (32Goppelt-Struebe M. Fickel S. Reiser C.O.A. Biochem. J. 2000; 349(Prt 1): 217-224Crossref Google Scholar). Interference with p42/44 MAP kinase activation led to an almost complete inhibition of Cox-2 and Egr-1 expression (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar, 25Goppelt-Struebe M. Stroebel M. J. Cell. Physiol. 1998; 175: 341-347Crossref PubMed Scopus (34) Google Scholar). Activation of p42/44 MAP kinases did not contribute significantly to LPA- or 5-HT-mediated CTGF expression, consistent with signaling pathways different from those leading to Cox-2 orEgr-1 expression. Induction of Cyr61, a protein closely related to CTGF, has recently been reported to be differentially sensitive to PD-98059 inhibition depending on the stimulus used (39Pendurthi U.R. Allen K.E. Ezban M. Rao V.M. J. Biol. Chem. 2000; 275: 14632-14641Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Whether p42/44 MAP kinase may also be involved in CTGFinduction under certain conditions remains to be investigated. The small GTP-binding protein RhoA is a downstream signaling molecule of LPA in many cell types (e.g. Refs. 33Mackay D.J.G. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar and 34Seasholtz T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (206) Google Scholar). Inhibition of Rho proteins by toxin B resulted in a concentration-dependent complete suppression ofCTGF mRNA expression. This effect was not restricted to LPA-mediated CTGF expression but was also observed when the cells were stimulated with 5-HT or TGF-β. Compared withCox-2 or Egr-1 mRNA expression, induction of CTGF was particularly sensitive to toxin B treatment, possibly related to the different signaling pathways activated. Basal expression ofCTGF, which was dependent on continuous transcriptional activity, as shown by the inducing effect of cycloheximide, was reduced to a similar extent. Concomitantly with the inhibition of CTGF mRNA expression, toxin B disrupted the actin cytoskeleton. Actin stress fibers, which are strongly expressed in mesangial cells culturedin vitro, were first dissolved and later appeared in a condensed form around the nucleus. Previous studies had shown that treatment with toxin B in the concentrations used did not lead to cell death by apoptosis or necrosis (23Reiser C.O.A. Lanz T. Hofmann F. Hofer G. Rupprecht H.D. Goppelt-Struebe M. Biochem. J. 1998; 330: 1107-1114Crossref PubMed Scopus (74) Google Scholar). Inhibition of Rho kinase, a downstream kinase of RhoA, implicated in RhoA-mediated actin polymerization (46Aspenstroem P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (286) Google Scholar), also impaired LPA-mediated CTGFexpression, hinting to a role for RhoA and stress fiber organization inCTGF induction. Direct disruption of the cytoskeleton by cytochalasin D also strongly affected the induction of CTGFby LPA and even more profoundly by TGF-β, indicating an important contribution of an intact cytoskeleton to signal transduction from the plasma membrane into the nucleus. Most of TGF-β signaling is mediated by Smad proteins which are activated in the cytosol and then translocate into the nucleus (reviewed in Ref. 47Piek E. Heldin C.-H. Ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (747) Google Scholar). Whether Smad translocation or activation is impaired by disruption of the actin cytoskeleton has not been reported yet. Furthermore, transcription ofCTGF by TGF-β is dependent on a novel response element interacting with unknown transcription factors (8Grotendorst G.R. Okochi H. Hayashi N. Cell Growth Differ. 1996; 7: 469-480PubMed Google Scholar). The steps of the TGF-β signaling cascade, which are dependent on an intact actin cytoskeleton, thus remain to be characterized. However, even complete disruption of the actin cytoskeleton, as observed after treatment of the cells with cytochalasin D, did not result in a reduction in CTGF mRNA expression comparable to the one brought about by toxin B, indicating specific effects of interference with Rho protein activation. Multiple target proteins of RhoA and the other members of the family, the involvement of which inCTGF mRNA expression is not excluded, have been described in different cell types, but mediators leading to gene expression have not yet been identified (34Seasholtz T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (206) Google Scholar). CTGF is a secreted protein, but due to its strong binding to heparin and other matrix components, it is detectable in the supernatants or as cell-associated protein depending on the cell type investigated (48Steffen C.L. Ball-Mirth D.K. Harding P.A. Bhattacharyya N. Pillai S. Brigstock D.R. Growth Factors. 1998; 15: 199-213Crossref PubMed Scopus (112) Google Scholar,49Boes M. Dake B.L. Booth B.A. Erondu N.E. Oh Y. Hwa V. Rosenfeld R. Bar R.S. Endocrinology. 1999; 140: 1575-1580Crossref PubMed Google Scholar). In a recent paper, Riser et al. (16Riser B.L. Denichilo M. Cortes P. Baker C. Grondin J.M. Yee J. Narins R.G. J. Am. Soc. Nephrol. 2000; 11: 25-38Crossref PubMed Scopus (21) Google Scholar) observed increased levels of CTGF protein in mesangial cell supernatants after treatment of the cells with heparin, in accordance with a high portion of cell-associated CTGF. These data are in accordance with our results, where CTGF protein was only detectable in cellular homogenates. In accordance with the increase in steady-state levels of CTGFmRNA, stimulation of mesangial cells with TGF-β or LPA also time-dependently increased CTGF protein. A single regulated band with an apparent molecular mass of 38 kDa was detected by the antibody used. To fully appreciate the dynamics of CTGF protein synthesis, i.e. secretion and degradation after stimulation with different types of mediators, more detailed studies will be necessary with antibodies, which are more defined with respect to the detection of proteolytic and potentially active CTGF fragments (50Brigstock D.R. Steffen C.L. Kim G.Y. Vegunta R.K. Diehl J.R. Harding P.A. J. Biol. Chem. 1997; 272: 20275-20282Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Taken together, induction of CTGF mRNA and protein in mesangial cells is not restricted to TGF-β as a stimulus but is stimulated by activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins. In the present study we showed activation by serotonin and LPA, but other activators of heptahelical receptors might also turn out to be regulators of CTGF expression thus extending the biological context of CTGF activation. The strong impact of the cytoskeletal organization on CTGF deserves further attention, because mesangial cells are contractile cells that change their phenotype during glomerular injury. The involvement of RhoA in the regulation of CTGF bears pathophysiologically and pharmacologically relevant implications: RhoA signaling is modulated by cGMP- and cAMP-dependent kinases (51Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. Chardin P. Pacaud P. Loirand G. J. Biol. Chem. 2000; 275: 21722-21729Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 52Lang P. Gesbert F. Delespine-Carmagnat M. Stancou R. Pouchelet M. Bertoglio J. EMBO J. 1996; 15: 510-519Crossref PubMed Scopus (481) Google Scholar, 53Dong J.M. Leung T. Manser E. Lim L. J. Biol. Chem. 1998; 273: 22554-22562Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), thus linking regulation of CTGF expression to activators of these pathways such as nitric oxide or activators of adenylyl cyclase. By interference with isoprenylation, the activity of Rho proteins is inhibited by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) (e.g. Ref. 54Essig M. Nguyen G. Prie D. Escoubet B. Sraer J.D. Friedlander G. Circ. Res. 1998; 83: 683-690Crossref PubMed Scopus (327) Google Scholar), and lovastatin and simvastatin are indeed potent inhibitors of CTGFinduction. 2M. Eberlein, J. Heusinger-Ribeiro, and M. Goppelt-Struebe, unpublished observation. RhoA, together with cytoskeletal alterations, thus seems to be an essential module of intracellular signaling pathways regulating the expression ofCTGF. The technical assistance of M. Rehm is highly acknowledged. The antibody directed against CTGF was kindly provided by S. Werner, Zurich, Switzerland, the cDNA directed againstCTGF by N. Wahab, London, UK, and toxin B by Drs. F. Hofmann and K. Aktories, Freiburg, Germany.

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