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Originally published In Press as doi:10.1074/jbc.M000976200 on September 6, 2000

J. Biol. Chem., Vol. 275, Issue 48, 37429-37435, December 1, 2000
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Induction of Connective Tissue Growth Factor by Activation of Heptahelical Receptors

MODULATION BY Rho PROTEINS AND THE ACTIN CYTOSKELETON*

Angelika HahnDagger, Juliane Heusinger-RibeiroDagger, Thomas Lanz, Susanne Zenkel, and Margarete Goppelt-Struebe§

From the Medizinische Klinik IV, Universität Erlangen-Nürnberg, Loschgestrasse 8, D-91054 Erlangen, Germany

Received for publication, February 7, 2000, and in revised form, August 31, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 of CTGF by transforming growth factor beta (TGF-beta ) 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-mediated CTGF induction was due to activation of 5-HT2A receptors 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-beta . Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction of CTGF by LPA and TGF-beta . 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 stimulated CTGF 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 CTGF expression 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 of CTGF expression induced by LPA and 5-HT, and also by TGF-beta .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1), and is also classified as a member of the CCN (CYR61, CTGF, and NOV) family (2, 3). 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 (4). 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 (5-7). In fibroblasts, it is most potently induced by transforming growth factor beta (TGF-beta ) (8). It stimulates fibroblast cell proliferation and mediates TGF-beta -induced anchorage-independent growth (9). Furthermore, CTGF is a potent stimulator of extracellular matrix synthesis (10, 11).

Elevated levels of CTGF are found in fibrotic lesions (e.g. Refs. 12-14) 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 (15). 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 of CTGF mRNA, which are further increased by TGF-beta (16). In accordance with elevated CTGF expression in diabetic glomerulosclerosis (15, 16), elevation of glucose levels enhanced CTGF mRNA levels in cultured mesangial cells (5). Up-regulation of CTGF by glucose was blocked by anti-TGF-beta antibodies, confirming CTGF as a downstream target of TGF-beta in mesangial cells (16). Besides TGF-beta , CTGF itself was able to induce its own mRNA expression (16). Mesangial cells are thus target cells of CTGF, as also shown by the induction of extracellular matrix proteins (fibronectin and collagen I and IV) (5). 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 (17). This prompted us to further investigate the regulation of CTGF mRNA 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 (18). Activated platelets are an abundant source of LPA, and high levels of the lysophospholipid (2-20 µM) are detectable in serum (19). 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 (18). Treatment of mesangial cells with LPA led to contraction of the cells (20) and stimulated proliferation (21, 22). Proliferation was shown to be mediated by the induction of the expression of the immediate early gene Egr-1 (23). Likewise, Cox-2, another example of an early response gene, was rapidly induced by LPA in mesangial cells (23). 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) (24, 25). 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 or Egr-1 (23, 26). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human TGF-beta 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.

Cell Culture-- Rat mesangial cells were isolated as described (27) and were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM L-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-- Northern blot analysis was performed as described previously (24). After stimulation for the indicated times, total RNA was extracted according to the protocol of Chomczynski and Sacchi (28) 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 (29). A cDNA specific for CTGF (full-length cDNA of human CTGF) was kindly provided by N. Wahab, London, UK (30). 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 to GAPDH or 18 S rRNA expression. The two-sided Student's t test for paired samples was used to calculate significant differences.

Western Blot Analysis-- 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 (31).

Staining of Actin Filaments-- 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Induction of CTGF mRNA Expression by Activation of Heptahelical Transmembrane Receptors-- TGF-beta has been characterized as a potent inducer of CTGF in different cell types, among them human mesangial cells (5). This was confirmed when rat mesangial cells were treated with TGF-beta with subsequent analysis of CTGF mRNA expression (Fig. 1A). 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. 1B). 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. 1C). Increased proliferation of mesangial cells and the induction of the early response genes Egr-1 and Cox-2 were observed in the same concentration range of LPA (23). LPA concentrations were within the range reported to occur in serum (2-20 µM (19)). Higher concentrations were not used to avoid nonspecific effects of the lysophospholipid. When mesangial cells were incubated with LPA plus TGF-beta , the increase in CTGF mRNA expression was additive (Fig. 1D). Low concentrations of LPA, which by themselves did not induce CTGF expression, did not significantly augment TGF-beta -mediated induction of CTGF. Additivity was also observed with TGF-beta 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. 8), LPA-mediated induction of CTGF was not inhibited in the presence of cycloheximide but strongly increased (Fig. 1E).


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  		Fig. 1.   Induction of CTGF mRNA. A, mesangial cells were treated with medium (C) or stimulated with LPA (L, 10 µM), serotonin (S, 1 µM), or TGF-beta 1 (T, 5 ng/ml) for the times indicated. CTGF mRNA expression was detected by Northern blot analysis. B, mesangial cells were incubated with LPA (10 µM) for the times indicated. As a control for equal loading of the gels, the ratio of CTGF expression and 18 S rRNA or GAPDH was used. To compare the mRNA expression of different experiments, expression of control cells was set to 0 and expression after 60 min of stimulation was set to 100%. Data are means ± S.D. of five experiments. C, mesangial cells were incubated with different concentrations of LPA for 2 h. CTGF expression after stimulation with 25 µM LPA was set to 100%. Data are means ± half range of two experiments. D, mesangial cells were incubated with different concentrations of LPA in the presence or absence of 5 ng/ml TGF-beta for 2 h. E, mesangial cells were preincubated with cycloheximide (CHX, 10 µg/ml) for 3 h and then analyzed (3 h). They were further incubated with or without LPA (10 µM) or CHX as indicated for 90 min (3 h + 90 min).

Association of CTGF Protein with Mesangial Cells-- 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-beta 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 (31) revealed the time-dependent induction of a protein of about 38 kDa when the cells were treated with LPA or with TGF-beta (Fig. 2A). 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 (16). 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. 2B). The time course of protein expression corresponded to the time course of mRNA induction. Co-incubation of mesangial cells with LPA and TGF-beta resulted in an increased expression of CTGF protein that was additive rather than synergistic (Fig. 2C). A second nonspecific band of a protein of about 33 kDa was detected on most blots, but with varying intensity (see also 5D).


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  		Fig. 2.   Induction of CTGF protein by LPA and TGF-beta . A, mesangial cells were treated with LPA (10 µM) or TGF-beta (5 ng/ml) for the times indicated (h). CTGF protein was detected in the cellular homogenates by Western blot analysis with an antibody directed against mouse CTGF. B, to quantify the protein expression the densitometric values of CTGF at 38 kDa were corrected for equal loading of the gels and blotting efficiency by densitometric values of the upper band, which was not regulated. To compare different experiments, expression of CTGF in control cells was set to 100%. Data are means ± S.D. of three experiments. *, p < 0.05 compared with control cells, Student's t test for paired samples. C, mesangial cells were stimulated for 3 h with LPA (L, 10 µM) or TGF-beta (T, 5 ng/ml) or a combination of both for 3 h. CTGF protein was detected in the cellular homogenates by Western blot analysis. C, control cells. The blot is representative of two experiments with similar results.

Differential Signaling Pathways for the Induction of CTGF and Cox-2-- As described before (23) expression of LPA-mediated Cox-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. 3A). 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. 3C). Co-incubation of mesangial cells with 5-HT and LPA did not further enhance CTGF expression, whereas Cox-2 expression was increased (Fig. 3A and Ref. 23).


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  		Fig. 3.   Pertussis-toxin insensitive induction of CTGF mRNA expression. A, mesangial cells were preincubated with PTX (100 ng/ml) for 18 h and then incubated with or without LPA (L, 10 µM) or 5-HT (S, 1 µM) for 1 h. Steady-state levels of CTGF and Cox-2 were determined by Northern blot hybridization. B, mesangial cells were treated as in A. To compare different experiments, the expression of CTGF after stimulation with 5-HT was set to 100%. Data are means ± S.D. of four experiments with 5-HT and means ± half range of two experiments with LPA. C, mesangial cells were preincubated with ketanserin for 60 min (Ket, 10 µM) and then further incubated with 5-HT for 1 h. +*, incubation with 5-HT in the presence of the solvent Me2SO. The blot is representative of two experiments with the same result.

LPA and 5-HT, both activate p42/44 mitogen-activated protein (MAP) kinases in mesangial cells (26, 32). 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. 4A and Refs. 23 and 25). 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 (32). 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 CTGF by LPA or 5-HT, however, was not impaired by PD-98059 and, thus, was independent of p42/44 MAP kinase activation.


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  		Fig. 4.   MAP kinase-independent induction of CTGF. A, mesangial cells were preincubated with PD-98059 (10 and 20 µM, PD10 and PD20) for 30 min. Then the cells were further incubated with LPA (10 µM) for 1 h. Northern blot analysis was used to detect CTGF and Cox-2 expression. B, mesangial cells were preincubated with PD-98059 as indicated (PD, 10 µM) for 30 min and then further incubated with LPA (10 µM) or 5-HT (1 µM) for 1 h. To compare different experiments, expression of CTGF after stimulation with LPA and 5-HT, respectively, was set to 100%. Data are means of three experiments

Role of Rho Proteins in CTGF Induction-- Rho proteins have been characterized as downstream mediators of LPA signaling in many cellular systems (e.g. Refs. 33 and 34). 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. 5A). Reduction of CTGF 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 of CTGF was sensitive to treatment with toxin B (Fig. 5B). Induction of the early response gene Cox-2 was also reduced but to a lesser extent (Fig. 5, A and B). 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 (35). The inhibitor reduced LPA-mediated induction of CTGF by about 50% (51.8 ± 16.0, means ± S.D., n = 3, p < 0.05; Fig. 5C). It also interfered with TGF-beta signaling, as did toxin B (see below). Inhibition of CTGF expression by toxin B was also observed at the protein level (Fig. 5D).


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  		Fig. 5.   Involvement of Rho proteins in CTGF regulation. A, mesangial cells were preincubated with toxin B for 3 h at the concentrations indicated. Then the cells were stimulated with LPA (10 µM) for 1 h. The blot is representative of four experiments with similar results. B, mesangial cells were pretreated with toxin B for 3 h at the concentrations indicated. Then the cells were stimulated with 5-HT (1 µM) for 1 h. Expression of CTGF and Cox-2 mRNA was detected by Northern blot analysis. The blot is representative of three experiments with similar results. C, mesangial cells were preincubated with Y-27632 (10 µM) for 1 h and then stimulated with LPA (L, 10 M) for 1 h and with TGF-beta (T, 5 ng/ml) for 4 h. D, mesangial cells were preincubated with toxin B (ToxB, 5 ng/ml) for 1 h and then stimulated with LPA plus TGF-beta (L/T, 10 µM and 5 ng/ml) for 3 h. CTGF protein expression was detected in cellular homogenates by Western blot analysis.

Role of the Actin Cytoskeleton in CTGF Induction-- 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-beta - or LPA-mediated induction of CTGF, 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 CTGF expression when the concentration of cytochalasin D was increased from 1 to 2 µg/ml (data not shown).


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  		Fig. 6.   Modulation of actin stress fibers in mesangial cells by toxin B and cytochalasin D. Mesangial cells were treated with toxin B (ToxB, 5 ng/ml) or cytochalasin D (CytoD, 1 µg/ml) for 1 or 3 h. Two examples of controls cells (Co) are shown in the upper row. Actin fibers were visualized by staining with rhodamine-phalloidin.


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  		Fig. 7.   Interference 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-beta for 4 h. B, to compare the CTGF expression in different experiments, expression of CTGF after stimulation with LPA after 1 h or TGF-beta after 4 h was set to 100%. Data are means ± S.D. of four (LPA) and three (TGF-beta ) experiments. Inhibition was significant with p < 0.05 (*); Student's t test for paired samples was used.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (36, 37). Furthermore, 5-HT has been related to increased matrix production in mesangial cells by induction of TGF-beta and enhanced synthesis of type IV collagen (38). Induction of CTGF 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-beta , but was also induced in an autocrine manner by recombinant CTGF (16). The data presented characterize activation of pertussis toxin-insensitive heptahelical receptors by LPA and 5-HT as novel signaling pathway to mediate CTGF induction. 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 (39), whereas des-Arg10-kallidin augmented CTGF mRNA levels more slowly, due to message stabilization (40). Activation of heptahelical receptors may thus differentially affect CTGF expression, 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. 41 or 42 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 (25, 43, 44). 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 or Cox-2 was pertussis toxin-sensitive in mesangial cells (23) as was c-fos induction in fibroblasts (45), 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 (32). Interference with p42/44 MAP kinase activation led to an almost complete inhibition of Cox-2 and Egr-1 expression (23, 25). 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 or Egr-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 (39). Whether p42/44 MAP kinase may also be involved in CTGF induction 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. 33 and 34). Inhibition of Rho proteins by toxin B resulted in a concentration-dependent complete suppression of CTGF 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-beta . Compared with Cox-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 of CTGF, 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 cultured in 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 (23). Inhibition of Rho kinase, a downstream kinase of RhoA, implicated in RhoA-mediated actin polymerization (46), also impaired LPA-mediated CTGF expression, hinting to a role for RhoA and stress fiber organization in CTGF induction. Direct disruption of the cytoskeleton by cytochalasin D also strongly affected the induction of CTGF by LPA and even more profoundly by TGF-beta , indicating an important contribution of an intact cytoskeleton to signal transduction from the plasma membrane into the nucleus. Most of TGF-beta signaling is mediated by Smad proteins which are activated in the cytosol and then translocate into the nucleus (reviewed in Ref. 47). Whether Smad translocation or activation is impaired by disruption of the actin cytoskeleton has not been reported yet. Furthermore, transcription of CTGF by TGF-beta is dependent on a novel response element interacting with unknown transcription factors (8). The steps of the TGF-beta 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 in CTGF mRNA expression is not excluded, have been described in different cell types, but mediators leading to gene expression have not yet been identified (34).

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 (48, 49). In a recent paper, Riser et al. (16) 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 CTGF mRNA, stimulation of mesangial cells with TGF-beta 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 (50).

Taken together, induction of CTGF mRNA and protein in mesangial cells is not restricted to TGF-beta 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 (51-53), 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. 54), and lovastatin and simvastatin are indeed potent inhibitors of CTGF induction.2 RhoA, together with cytoskeletal alterations, thus seems to be an essential module of intracellular signaling pathways regulating the expression of CTGF.

    ACKNOWLEDGEMENTS

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 against CTGF by N. Wahab, London, UK, and toxin B by Drs. F. Hofmann and K. Aktories, Freiburg, Germany.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, Go 413-8 and SFB 423, B3.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.

Dagger Both authors contributed equally to this manuscript.

§ To whom correspondence should be addressed: Tel.: 49-9131-853-9201; Fax: 49-9131-853-9202; E-mail: Goppelt-Struebe@rzmail.uni-erlangen.de.

Published, JBC Papers in Press, September 6, 2000, DOI 10.1074/jbc.M000976200

2 M. Eberlein, J. Heusinger-Ribeiro, and M. Goppelt-Struebe, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: CTGF, connective tissue growth factor; Cox, cyclooxygenase; LPA, lysophosphatidic acid; MAP kinase, mitogen-activated protein kinase; 5-HT, serotonin; TGF-beta , transforming growth factor beta ; PTX, pertussis toxin; FCS, fetal calf serum; MOPS, 4-morpholinepropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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