Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor Trka

Connective Tissue Growth Factor CCN2 Interacts with and Activates the Tyrosine Kinase Receptor TrkA

Nadia Abdel Wahab, Benjamin S. Weston, and Roger M. Mason Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, South Kensington, London, United Kingdom

Connective tissue growth factor (CTGF) is implicated as a factor promoting tissue fibrosis in several disorders, including diabetic nephropathy. However, the molecular mechanism(s) by which it functions is not known. CTGF rapidly activates several intracellular signaling molecules in human mesangial cells (HMC), including extracellular signal–related kinase 1/2, ␣ ␦ Jun NH2-terminal kinase, protein kinase B, CaMK II, protein kinase C , and protein kinase C , suggesting that it functions via a signaling receptor. Treating HMC with CTGF stimulated tyrosine phosphorylation of proteins 75 to 80 and 140 to 180 kD within 10 min, and Western blot analysis of anti-phosphotyrosine immunoprecipitates identified the neurotrophin receptor -TrkA (molecular weight approximately 140 kD). Cross-linking rCTGF to cell surface proteins with 3,3؅-dithiobis(sulfosuccin imidylpropionate) revealed that complexes formed with TrkA and with the general neurotrophin co-receptor p75NTR. rCTGF stimulated phosphorylation of TrkA (tyr 490, 674/675). K252a, a known selective inhibitor of Trk, blocked this phosphorylation, CTGF-induced activation of signaling proteins, and CTGF-dependent induction of the transcription factor TGF-␤– inducible early gene in HMC. It is concluded that TrkA serves as a tyrosine kinase receptor for CTGF. J Am Soc Nephrol 16: 340–351, 2005. doi: 10.1681/ASN.2003100905

onnective tissue growth factor (CTGF; CCN2) is a Recently, we proposed that CTGF directly enhances the TGF- 38-kD secreted protein with multiple domains, en- ␤/Smad signaling pathway. The mechanism underlying this C coded by an immediate-early gene, and is a member seems to be via the rapid induction of the transcription factor of the CCN protein family (1). An increasing number of TGF-␤–inducible early gene (TIEG) (11). TIEG has been shown to studies in the past few years indicate that CTGF is strongly bind to the promoter of the Smad7 gene and represses its tran- implicated in the pathogenesis of a variety of fibrotic disor- scription (12). Because Smad7 is a potent inhibitory Smad, it seems ders, including diabetic nephropathy (2). Functionally, the that CTGF simply blocks the negative feedback loop of the TGF-␤ growth factor has been shown to regulate many aspects of signaling pathway, allowing its continued activation. The rapid cell behavior, including cell adhesion, migration, growth, activation of several intracellular signaling pathways by CTGF, chemotaxis, and the synthesis and accumulation of matrix together with its ability to induce the expression of several genes proteins (3). However, the molecular mechanism(s) by which in mesangial cells, including the early gene TIEG, suggested that it functions is not clear. The presence of multiple domains in a specific signaling receptor must exist. Previous cross-linking CTGF suggests a role for the growth factor as an integrator of studies revealed CTGF-receptor complexes with an apparent mo- several other signaling molecules such as growth factors, lecular weight of 280 kD, present in chondrocytes, osteoblasts, and integrins, and extracellular matrix proteins. CTGF has been endothelial cells (13). CTGF was also found to bind to LDL recep- reported to bind directly bone morphogenic protein–4 and tor–related protein. However, this may facilitate clearance rather ␤ TGF- through its von Willebrand type C domain, leading to than signal transduction (14). inhibition of bone morphogenic protein and enhancement of In the present report, we provide for the first time evidence ␤ TGF- signaling (4). CTGF also has been shown to bind to that CTGF interacts with TrkA and p75NTR, a dual-receptor integrins (5–10), and this interaction may mediate some of system that is known to transduce neurotrophin signals. There the cellular phenomena mentioned above. are three Trk receptor tyrosine kinase genes (TrkA, TrkB, and TrkC) and a single gene encoding the neurotrophin receptor, p75NTR. On ligand binding, Trk dimerize and autophosphory- Received October 31, 2003. Accepted October 25, 2004. late, leading to the activation of several small G proteins, in- Published online ahead of print. Publication date available at www.jasn.org. cluding Ras, Rap-1, and the Cdc 42-Rac-Rho family, as well as Address correspondence to: Dr. Nadia Wahab, Renal Section, Division of Med- of pathways regulated by mitogen activated protein kinase icine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, (MAPK), phosphatidylinositol 3-kinase, and phospholipase C-␥ ϩ ϩ W12 ONN, UK. Phone: 44-20-838-32718; Fax: 44-20-838-32062; E-Mail: (15). On activation, Trk also promote a rapid increase in cyto- [email protected] plasmic calcium level. This seems to arise from both release of The current affiliation for N.A.W. and R.M.M. is Renal Section, Division of Medicine, Imperial College London, Hammersmith Hospital, London, United intracellular stores and uptake of extracellular calcium (16,17). Kingdom. Moreover, activated Trk interact, directly or indirectly, with

many cytoplasmic adaptor proteins, leading to a variety of nant CTGF (CTGF/V5 fusion protein) was expressed in transformed biologic responses, including, cell proliferation and survival; HMC and purified from the medium using Talon metal affinity resin, axonal and dendritic growth and remodeling; assembly and as reported previously (2). Alternatively, r-CTGF (nonfusion protein) remodeling of cytoskeleton; membrane trafficking and fusion; was expressed in the baculovirus system and was a gift from FibroGen Inc. (South San Francisco, CA). Rabbit anti-CTGF (pAb2) and chicken and synapse formation, function, and plasticity (18). NTR anti-CTGF (pIgY3) were also supplied by FibroGen Inc. Phosphothioate The pan-neurotrophin receptor p75 is a member of a antisense and control oligonucleotides directed to TrkA (GTGAAGAT- superfamily that includes the TNF, B cell antigen CD40, and Fas GAAGCTGGT; ACTACTACACTAGACTAC) and p75NTR (TTCTGCT- NTR receptors (19). p75 signals via pathways involved with ac- TGTCCTG; GCTCTATGACTCCCAG) were designed and manufac- tivation of sphingomyelinase and ceramide production (20), tured by Biognostik GmbH (Go¨ttingen, Germany), who own the ␬ NF- B (21), and Jun NH2-terminal kinase (JNK) (22). Although intellectual property rights to the sequences. both receptors mediate the biologic effects of neurotrophins, it seems that Trk play the central role in signaling. However, the contribution of p75NTR is not fully understood. It has been Cross-Linking and Membrane Preparation proposed that p75NTR can act as a co-receptor with Trk to create Cell layers were washed twice with cold binding buffer (PBS and 0.5% glucose) and incubated with CTGF in binding buffer for2hat4°C. high-affinity receptors for different neurotrophins (23) either by After incubation, the cell layers were washed five times with cold presenting the ligand to the Trk receptor, or by altering the binding buffer and incubated with 1 mM 3,3Ј-dithiobis(sulfosuccinimi- conformation of the Trk receptors through allosteric interaction dylpropionate) (DTSSP) or disuccinimidyl suberate (Pierce Biotechnol- (24). It can also function on its own (25). Yet another interesting ogy, Tattenhall, Cheshire, UK) in PBS for 30 min at room temperature. NTR finding is that p75 can function as a receptor for the imma- The reaction was quenched for 15 min at room temperature by the ture unprocessed forms of neurotrophins, whereas Trk func- addition of 50 mM Tris buffer (pH 7.5). Cell layers were washed with tions as a receptor for the mature forms (26). Cross-talk be- wash buffer (10 mM Tris buffer [pH 7.5], 5 mM MgCl, and 150 mM tween the Trk and other membrane receptors, such as G NaCl), scraped in homogenizing buffer (10 mM Tris buffer [pH 7.5], 250 ϫ protein–coupled receptor, vannilloid receptor, and c-Ret, have mM sucrose, 1 mM EDTA, 5 mM MgCl, 150 mM NaCl, and 1 protease inhibitor cocktail; Roche Applied Science, Mannheim, Germany), also been reported (18). passed through a 25-G needle, and homogenized on ice with 30 to 40 Ligand engagement stimulates the endocytosis of the ligand– cycles in a Dounce homogenizer. The homogenate was centrifuged for Trk complex into vesicles via a clathrin-dependent mechanism 10 min at 2500 ϫ g at 4°C. The resulting supernatant was centrifuged (27). The receptors remain catalytically active within the sorting for 90 min at 45,000 ϫ g at 4°C. The membrane-enriched pellet was vesicles, which are called “signaling endosomes” (28). Inhibit- solubilized for 1 h in solubilizing buffer (10 mM Tris buffer [pH 7.5], 5 ing endocytosis inhibits extracellular signal–related kinase mM MgCl, 150 mM NaCl, 1% Triton-X100, and 1ϫ protease inhibitor (ERK) 1/2 activation in response to neurotrophin stimulation cocktail). Soluble membrane proteins were collected after further cen- (29). trifugation for1hat45,000 ϫ g at 4°C. When rCTGF/V5 fusion protein As Trk receptors have been found to bind a large number of was used, CTGF cross-linked proteins were either immunoprecipitated with rabbit anti-CTGF antibody or captured on a Pull-Down PolyHis adaptor proteins and multiple intracellular signaling pathways column (Pierce). When rCTGF was used, CTGF cross-linked proteins are activated by them, as well as modulated by p75NTR, this were captured on a goat anti–CTGF-C terminal domain–sepharose may explain the multifunctional properties of CTGF. The re- immunoaffinity column, using an IgG-sepharose column as a control sults presented in this study show that Trk tyrosine kinase (FibroGen Inc.). After extensive washing of the columns with solubi- activity is required for the CTGF-dependent induction of the lizing buffer, bound proteins were solubilized in reducing SDS-PAGE transcription factor TIEG. loading buffer, boiled for 5 min, and resolved on 4 to 12% gradient gels by SDS-PAGE. Gels were either stained with Coomassie blue or used for Western blotting. Materials and Methods Cell Cultures, Antibodies, and Reagents Primary normal adult human mesangial cells (HMC; CC-2259, lot RNA Extraction and Reverse Transcriptase–PCR Analysis 3F1510) were purchased from BioWhittaker (Wokingham, Berkshire, Total RNA was extracted from 6 ϫ 106 mesangial cells using the UK), maintained in culture as described previously (2), and used at RNAzol B method (AMS Biotechnology [UK] Ltd., Oxfordshire, UK). passages 9 to 10. Phospho-Akt (P-Ser 472/473/474) antibody was from Equal amounts of total RNA (2 ␮g) from each sample were reverse- ϩ Pharmingen (San Diego, CA). Phospho-Akt (P-Thr 308) and ERK5 transcribed into cDNA using SuperScript II RNase H reverse tran- antibodies were from Sigma (Gillingham, Dorset, UK). Phospho- scriptase (Life Technologies BRL, Paisley, Scotland, UK) and random ERK1/2 pathway sampler, phospho-JNK pathway sampler, phospho primers. Equal amounts (0.5 ␮l) of the reverse transcription reaction (20 P38 MAPK pathway sampler, phospho-PKC␦, phospho-PKC␣, phos- ␮l) were subjected to PCR amplification in a 100-␮l volume that con- pho-TrkA (Tyr674/675), and phospho-TrkA (Tyr490) antibodies were tained 10 ␮lof10ϫ PCR buffer, 16 ␮l of dNTP (1.25 mM each), 2 mM ␮ from New England BioLabs (Hitchen, Herts, UK). Phospho-CaMKII MgCl2, 5 M betaine (Sigma), 0.5 M of each specific primer, and 1.25 U (P-Thr286) antibody was from Promega (Southampton, Hants, UK), of Amplitaq DNA polymerase (Life Technologies BRL). Amplification and anti-phosphotyrosine antibody was from Santa Cruz (Autogen was started with 5 min of denaturation at 94°C followed by 30 PCR Bioclear, Calne, Wilts, UK). Anti-TrkA antibody was obtained from cycles for all genes. Each cycle consisted of 60 s at 94°C, 60 s at 55°C, Upstate Biotechnology (Milton Keynes, UK). Anti–TIEG-1 antibody and 60 s at 72°C. The final extension was for 10 min at 72°C. A control was a gift from Dr. Steven Johnson (Mayo Foundation, Rochester, MN). omitting the reverse transcriptase step was also performed. The se- K-252a was purchased from Calbiochem (Nottingham, UK). Recombi- quences of primers were as described by Anderson et al. (30). 342 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 340–351, 2005

Western Blotting ysis using an anti-phosphotyrosine antibody. Short-term expo- Cells were lysed in reducing SDS-PAGE loading buffer and imme- sure of the blot (Ͻ5 s) showed that CTGF fusion protein (40 diately scraped off the plate. Cell lysates were sonicated for 10 s to ng/ml) stimulates the tyrosine phosphorylation within 10 min shear DNA. Samples were boiled for 5 min and resolved on 4 to 12% of at least two major proteins with apparent molecular weight gradient gels by SDS-PAGE. Proteins were transferred onto a polyvi- of approximately 75 to 80 and 140 to 150 kD in HMC (Figure 2). nylidene difluoride membrane filter (Immobilin-P; Millipore, Bedford, Another phosphotyrosine protein (molecular weight 45 kD) UK) using a BioRad transfer apparatus. Blots were incubated in block- was detected in control cell lysate and seems to be reduced in ing buffer that contained 1ϫ TBS and 0.1% Tween-20 with 5% (wt/vol) nonfat dry milk for 1 h. Immunodetection was performed by incubating response to the CTGF treatment. the blots in primary antibody at the appropriate dilution in antibody dilution buffer (1ϫ TBS and 0.1% Tween-20 with 5% BSA) overnight at CTGF Interacts with HMC Surface Proteins 4°C. Blots then were washed three times with washing buffer (1ϫ TBS To investigate whether CTGF interacts with HMC surface and 0.1% Tween-20) and incubated with secondary horseradish peroxi- proteins, we allowed CTGF to bind to the cell surface, per- dase–conjugated antibodies for1hatroom temperature. Bound anti- formed a subsequent cross-linking procedure, and isolated a bodies were visualized using the enhanced chemiluminescence reagent membrane-enriched fraction from the cells. After solubilization, Luminol (Autogen Bioclear UK Ltd, Wiltshire, UK). Prestained molec- this was immunoprecipitated with a rabbit anti-CTGF anti- ular weight standards (Amersham International PLC, Amersham, UK) body. Covalently linked CTGF complexes then were analyzed were used to monitor protein migration. by PAGE and Western blotting with a chicken anti-CTGF antibody. As shown in Figure 3, lane 2, CTGF seems to be cross- Immunofluorescence Staining linked with membrane proteins to form complexes of apparent Cells were fixed with 3.7% paraformaldehyde and permeabilized molecular weight 180 kD and Ͼ220 kD, the latter being a large with 0.5% Triton X-100 in PBS for 10 min at room temperature. Cov- diffuse band. These complexes were not immunoprecipitated erslips then were incubated overnight at 4°C with serum (5% in PBS) from the membrane-enriched fraction when the cross-linking from the same species as that in which the secondary antibody was raised. After this, they were incubated with primary antibodies (at step was eliminated (lane 1). optimum dilution in PBS that contained 3% BSA) for1hat37°C. To ascertain whether CTGF activates an RTK, we incubated Coverslips then were washed and incubated in the dark for 1 h with serum-starved HMC in the presence or absence of CTGF for 15 fluorescein-conjugated secondary antibody (Sigma Aldrich, Dorset, min, lysed the cells, and immunoprecipitated phosphotyrosine UK). After staining, the coverslips were mounted on glass slides with proteins. The immunoprecipitated proteins were analyzed by antifade mounting media (Vector Labs, Peterborough, UK) and exam- Western blotting using antibodies against TrkA (Figure 4A, B, ined using a fluorescence microscope. C, and E) and EGF receptor (EGFR; data not shown), known tyrosine kinase receptors. Only the anti-TrkA antibody cross- Results reacted strongly with a band of approximately 140 kD (Figure CTGF Activates Several Intracellular Signaling Pathways 4A). The intensity of this band was stronger when cells were To understand the molecular mechanisms by which CTGF incubated with CTGF (lane 2), indicating its activation by the functions, we used purified rCTGF-V5 fusion protein to iden- growth factor. The interaction of CTGF with the TrkA receptor tify the intracellular signal pathways that are activated in re- was confirmed by different experiments in which either His- sponse to the growth factor in HMC. We found that CTGF tagged CTGF/V5 fusion protein or rCTGF expressed in the rapidly triggers the activation of the classical MAPK (ERK1/2) baculovirus system was allowed to bind to the cell surface and and JNK pathways (Figure 1, A and B) but not the p38 MAPK then cross-linked to its ligand(s) using the reversible cross- (data not shown). The figure shows maximal activation of these linker DTSSP. The latter is cleaved by reducing agents. Subse- kinases after 15 min of CTGF stimulation. CTGF stimulation quently, a membrane fraction was prepared and cross-linked also led to the activation of Akt, also known as protein kinase CTGF complexes were captured on affinity metal beads or on B (PKB), at both the known phosphorylation sites: thr-308 and anti–C-terminus CTGF antibody affinity beads. The captured ser-473 (Figure 1C). It is interesting that the activation of thr-308 complexes were subjected to SDS-PAGE under reducing con- (by a phosphoinositide-dependent kinase 1 whose activity is ditions and analyzed by Western blots. The results in Figure 4, strictly dependent on 3-phosphorylated inositol lipids [31]) B through D, clearly indicate that CTGF interacts with the TrkA seems to be rapid and sustained in comparison with the acti- receptor. As Trk receptors have been shown to interact with the vation of ser-473. Phosphorylation of the latter (by integrin- pan neurotrophin receptor p75NTR, blots were stripped and linked kinase [32]) seems to be transient with a maximal level at reprobed using an anti-p75NTR antibody. As shown in Figure 15 min and return to a level close to the basal one within 30 min 4E, the antibody cross-reacted with a protein of the correct of CTGF exposure. CTGF stimulation also led to the transient molecular weight for P75NTR. Thus, our results indicate that activation of CaMK II (Figure 1C). Other kinases that seem to be CTGF interacts with TrkA and p75NTR, two receptors that are activated in response to CTGF are PKC␦ and PKC␣ (Figure 1D). known to be activated by the neurotrophin nerve growth factor These results indicate that CTGF signals through a receptor, (NGF). To confirm this interaction, MC were depleted of TrkA most probably a receptor tyrosine kinase (RTK), because RTK or p75NTR receptors using antisense oligonucleotides (2 ␮M for are commonly known to activate these kinases. To test this 3 d), as described previously by Wahab et al. (2). Equal amount hypothesis, we exposed HMC to CTGF for various periods of of r-CTGF were allowed to bind to the cell surface and then time, prepared cell lysates, and performed Western Blot anal- cross-linked with DTSSP as above. Membrane preparations J Am Soc Nephrol 16: 340–351, 2005 CTGF Activates the TrkA Receptor 343

Figure 1. Connective tissue growth factor (CTGF) activates intracellular signaling pathways. Serum-starved human mesangial cells (HMC) were incubated in the presence of CTGF/V5 fusion protein for the periods of time indicated, after which the cells were lysed. Equal amounts of lysate protein were subjected to SDS-PAGE and analyzed by Western blotting using phospho-specific antibodies against the constituent proteins of the mitogen-activated protein kinase (MAPK) pathway including the MEK1/2 and their downstream targets extracellular signal–related kinase 1 and 2 (ERK1/2) and p90-ribosomal-S6 kinase (p90RSK; A) Jun ␤ NH2-terminal kinase (JNK; B), and protein kinase B (PKB) and CamKII (C). -Actin is shown as a marker for equal protein loading. (D) Cells were grown on coverslips and serum-starved for 48 h before incubation in medium in the absence (a and c) or presence of 40 ng/ml CTGF-fusion protein (b and d) for 30 min. Cells were fixed, permeabilized, and probed with anti-phospho PKC␦ (a and b) and PKC␣ (c and d) primary antibodies and then with fluorescein-conjugated secondary antibody, as described in the Materials and Methods section. White arrows in b and d point to plasma membrane translocation of PKC␦ and PKC␣, characteristic of PKC enzyme activation. Results are representative of three separate experiments. 344 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 340–351, 2005

receptors interacting with CTGF (Figure 4, B and C). Both TrkA and p75NTR control antisense oligonucleotides did not effect the amount of the interacting receptors (compare lanes 3 and 5). Attempts to confirm chemically the identity of these receptors using MALDI-TOF-MS analysis were unsuccessful because of their relatively low abundance and, probably, high level of glycosylation of these receptors.

HMC Express Trk Receptors To investigate whether HMC express any members of the Trk, we extracted total RNA from HMC and performed reverse transcriptase–PCR analysis. The results (Figure 6) show that HMC express the three members of the neurotrophin receptor family—TrkA, TrkB, and TrkC—as well as the pan receptor Figure 2. CTGF induces tyrosine phosphorylation of different p75NTR. It is interesting that HMC also express the neurotro- proteins. Serum-starved HMC were incubated in the presence phin factors NGF and brain-derived growth factor. of 40 ng/ml CTGF/V5 fusion protein for the periods of time indicated, after which the cells were lysed. Equal amounts of lysate protein were subjected to SDS-PAGE and analyzed by CTGF Activates TrkA in HMC Western blotting using anti-phosphotyrosine antibody. Results On binding its ligand, TrkA autophosphorylates on several are representative of three separate experiments. tyrosine residues, leading to the association and activation of multiple effectors. Phosphorylation at Tyr490 is required for Shc association and activation of the Ras-MAPK cascade. Phos- phorylations at Tyr674/675 lie within the catalytic domain and reflect Trk kinase activity. Therefore, we tested whether stim- ulating cells with CTGF leads to the phosphorylation of TrkA at these residues. The results in Figure 7 show that CTGF induces an increase in the phosphorylation of the receptor at the Tyr490 and Tyr674/675 residues by 1.8-fold and 2.5-fold, respectively.

Inhibition of CTGF-Induced Signaling Pathways by K252a K252a is an alkaloid-like kinase inhibitor that is known to selectively inhibit Trk (33). K252a blocked the rapid phosphorylation of ERK1/2/JNK/ERK5 and PKB in HMC stimulated with CTGF (Figure 8), indicating that the phosphorylation of these kinases is indeed induced by ligand activation of the Trk receptor. Furthermore, treatment with K252a led to the accu- Figure 3. CTGF interacts with HMC surface proteins. CTGF/V5 mulation of TrkA at the plasma membrane of HMC that were fusion protein was allowed to bind to the cell surface and then exposed to CTGF (Figure 9). This indicates that internalization chemically cross-linked to its ligands with BS3, after which a and sorting of receptor–CTGF complexes was blocked as a membrane-enriched fraction was prepared from the cells, as described in the Materials and Methods section. Cross-linked result of inhibition of the receptor tyrosine kinase activity by CTGF complexes were immunoprecipitated using rabbit anti- K252a. This is consistent with the hypothesis that TrkA activa- CTGF antibody, resolved by SDS-PAGE, and analyzed by tion is an obligatory step in the recruitment of activated Trk Western blotting using chicken anti-CTGF antibody (lane 2). receptors to clathrin-coated pits and entry into the endocytic The cross-linking step was omitted for some cultures (lane 1). pathway (34). The results in Figure 9 also indicate that K252a Results are representative of three separate experiments. completely blocks TrkA phosphorylation at Tyr490 but is less effective in blocking phosphorylation at Tyr674/675. Tyr674/ 675 are within the catalytic domain of TrkA but are not asso- were made, and equal amounts of solubilized protein were ciated with signaling. Phosphorylation of these tyrosines might incubated with anti–C-terminus CTGF antibody affinity beads. turn over at a slower pace than those such as tyr 490, which are Bound complexes were subjected to SDS-PAGE under reducing associated with effector binding (35). Thus, tyr 674/675 may be conditions and analyzed by Western blots. Figure 5 shows that more readily detected in the presence of K252a than tyr 490. reducing the expression level of the TrkA receptor by treatment It is interesting that similar results were obtained with HMC with antisense oligonucleotide (lane 2) resulted in a reduction that were treated with 50 ng/ml NGF (Figure 10). The activa- in the amount of TrkA receptors that bound to CTGF (Figure tion of TrkA by both CTGF and NGF were confirmed by 2B), but it had no significant effect on the amount of p75NTR Western blot analysis, as shown in Figure 11. bound to CTGF (Figure 2C). In contrast, reducing the expres- For further evaluating the effect of K252a on TrkA autophos- sion level of p75NTR (lane 4) reduced the amount of both phorylation and signaling, HMC were serum starved for 24 h, J Am Soc Nephrol 16: 340–351, 2005 CTGF Activates the TrkA Receptor 345

Figure 4. CTGF interacts with TrkA and p75NTR in HMC. (A) Serum-starved HMC were incubated in the absence (lane 1) or presence (lane 2) of CTGF/V5 fusion protein (40 ng/ml) for 15 min, after which cell lysates were prepared in RIPA buffer. Equal amounts of lysate protein were immunoprecipitated using anti-phosphotyrosine beads. Bound proteins were resolved by SDS-PAGE and analyzed by Western blotting using an antibody against TrkA. (B) HMC were incubated in the absence (lane 1) or presence (lane 2) of His tagged-CTGF/V5 fusion protein (200 ng/ml) for2hat4°Ctoallow it to bind to cell surface receptors, after which it was chemically cross-linked with 3,3Ј-dithiobis(sulfosuccinimidylpropionate). A membrane-enriched fraction was prepared and solubilized. Equal amounts of solubilized protein were incubated with metal affinity beads. Bound proteins were subjected to SDS-PAGE under reducing conditions and Western blotting using an antibody against TrkA. (C) HMC were incubated with 200 ng/ml rCTGF (FibroGen Inc.) for2hat4°C. Bound CTGF was cross-linked as above, and a membrane- enriched fraction was prepared and solubilized. Cross-linked CTGF complexes in the solubilized fraction were captured on anti–C-terminus-CTGF antibody affinity beads (lane 1), or the fraction was incubated with control IgG affinity beads (lane 2). Bound proteins were analyzed by Western blotting using anti-TrkA antibody. (D) The sample shown in C, lane 1, was boiled for a longer time and then Western blotted using anti-TrkA antibody. The same blot (D) was stripped and reprobed using anti-p75NTR antibody (E). Results are representative of four separate experiments. pretreated with increasing concentrations of the inhibitor for 30 tant first to understand fully the precise role and the molecular min, and then stimulated with 40 ng/ml CTGF/V5 for 15 min. effector mechanisms of this growth factor. Cells were lysed and analyzed for phosphorylation of TrkA and In this report, we provide evidence that CTGF triggers the the downstream effector MAPK. Figure 12 shows that CTGF- rapid simultaneous activation of the signaling pathways MAPK induced total tyrosine phosphorylation of TrkA, phosphoryla- (ERK 1/2), JNK, ERK5, phosphatidylinositol 3-K, CaM-KII, tion at TrkA tyr-490 residue, and p-MAPK all are significantly PKC␣, and PKC␦ in HMC. By subsequent binding, cross-link- inhibited by K252a at 10-nM concentration. This result is in ing, and immunoprecipitation studies, we identified the ty- NTR good agreement with the published IC50 for Trk (approxi- rosine kinase receptor TrkA and p75 as two receptors that mately 3 nM) (33,36), whereas inhibitory effects on other ki- interact with CTGF. TrkA and p75NTR are known to be acti- nases require much higher concentrations (36). vated by the neurotrophin factor NGF. Neurotrophins are survival and differentiation factors in the nervous system, and TrkA Tyrosine Kinase Activity is Required for although both receptors mediate the biologic effects of neuro- CTGF-Dependent Induction of TIEG trophins, it seems that TrkA plays the central role in signaling, To determine whether Trk tyrosine kinase activity is required whereas the contribution of p75NTR is not fully understood. for CTGF-dependent induction of the transcription factor TIEG-1, we treated HMC with K252a 30 min before incubation Activation of TrkA in HMC in response to CTGF treatment was with CTGF for 2 h. As shown in Figure 13, CTGF enhanced the further supported by the ability of the Trk selective inhibitor expression of TIEG-1 by 3.4-fold (P Ͻ 0.0001). K252a was a K252a to block the rapid phosphorylation of ERK1/2, JNK, potent inhibitor for TIEG induction at a concentration that is PKB, and ERK5. known specifically to inhibit Trk kinase activity in other cells On binding and activation of Trk by neurotrophins, the ligand– (33,36). receptor complex is endocytosed into vesicles via a clathrin- dependent mechanism (27,29), where receptors remain catalyti- Discussion cally active within these vesicles (28). Inhibiting endocytosis with It is becoming clear that CTGF is implicated in the pathogen- specific inhibitors, or chemical inhibition of Trk kinase, inhibits esis of diabetic nephropathy and possibly of other fibrotic ERK1/2 activation in response to neurotrophin stimulation (29). disorders. Thus, therapeutic approaches that could selectively Previously, we demonstrated that CTGF is endocytosed from the block CTGF activity would be beneficial. However, it is impor- cell surface in endosomes, from which the growth factor translo- 346 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 340–351, 2005

Figure 7. CTGF activates TrkA in HMC. Serum-starved HMC were incubated in the absence (lane 1) or the presence (lane 2) of CTGF/V5 (40 ng/ml) for 15 min, after which the cells were lysed. Equal amounts of lysate protein were subjected to SDS- PAGE and analyzed by Western blotting. Blot A was probed with anti-TrkA antibody. Blot B was probed with anti–phos- Figure 5. Effect of TrkA and p75NTR antisense oligonucleotide pho-TrkA (Tyr490) antibody, whereas blot C was probed with treatment on receptor interaction with CTGF in HMC. HMC anti–phospho-TrkA (Tyr674/675). Results are representative of were treated or not treated with phosphothioate antisense or three separate experiments. control oligonucleotides directed to TrkA or p75NTR for3d, after which the cells were used to perform cross-linking experiments with r-CTGF, as described in Figure 4C. Equal amounts of solubilized membrane proteins were incubated with anti– C-terminus-CTGF antibody affinity beads. Bound proteins were analyzed by Western blotting using anti-TrkA and p75NTR antibodies (A). (B) Densitometric quantification of the TrkA blots. (C) Densitometric quantification of the p75NTR blots. Results (mean Ϯ SEM) represent three separate experiments (n ϭ 6). *P Ͼ 0.003; **P Ͼ 0.0001; ϩP Ͼ 0.007.

Figure 8. Inhibition of CTGF-induced signaling pathways by K252a. Serum-starved HMC were incubated with CTGF/V5 (40 ng/ml) in the presence or absence of K252a (100 nM) for the period of time indicated, after which the cells were lysed. Equal amounts of lysate protein were subjected to SDS-PAGE and Figure 6. HMC expresses Trk receptors. Total RNA was ex- analyzed by Western blotting using phospho-specific antibod- ␤ tracted from HMC and used for reverse transcriptase–PCR as ies against the indicated protein kinases. -Actin is shown as a described in the Materials and Methods section. After amplifi- marker for equal protein loading. cation, 10 ␮l of each PCR reaction product was electrophoresed through a 1.2% (wt/vol) agarose gel that contained ethidium bromide (0.5 ␮g/ml). Results are representative of three sepa- observed when the cells were treated with NGF. Activation of the rate experiments. ERK5 pathway in HMC that were treated with CTGF is consistent with its preferential activation within vesicles (38). Treatment of cates into the nucleus (37). In the present study, we showed that HMC with CTGF or NGF in the presence of K252a led to the the addition of CTGF to HMC induces the activation of TrkA and accumulation of TrkA at the plasma membrane. However, this its accumulation in vesicles (Figure 9), which were similar to those phenomenon was seen only when probing with the phospho-Trk J Am Soc Nephrol 16: 340–351, 2005 CTGF Activates the TrkA Receptor 347

Figure 9. Inhibition of CTGF-dependent activation of TrkA by K252a in HMC. HMC were grown on coverslips and serum-starved for 24 h, after which they were incubated with CTGF/V5 (40 ng/ml) in the presence or absence of K252a (100 nM) for 30 min. Cells were fixed, permeabilized, and immunostained using anti–phospho-TrkA (Tyr674/675) or (Tyr490) antibodies. Results are representative of three separate experiments.

Figure 10. Inhibition of nerve growth factor (NGF)-dependent activation of TrkA by K252a in HMC. HMC were grown on coverslips and serum-starved for 24 h, after which they were incubated with NGF (50 ng/ml) in the presence or absence of K252a (100 nM) for 30 min. Cells were fixed, permeabilized, and immunostained using anti–phospho-TrkA (Tyr674/675) or (Tyr490) antibodies.

(Tyr674/675) antibody, which is specific for phosphorylated ty- into the endocytic pathway (34). K252a also blocks the CTGF- rosine residues that lie within the catalytic domain. In contrast, the dependent induction of the transcription factor TIEG (Figure 11), phospho-Trk (Tyr490) antibody, which is specific for the tyrosine indicating that Trk tyrosine kinase activity is required for CTGF to residue required for Shc association and activation of the Ras- induce expression of this gene. MAPK cascade, did not show accumulation of phospho-TrkA at The principal domains in Trk that determine affinity and the plasma membrane. This suggests that K252a is more potent specificity of binding of neurotrophins are the two IgC2 do- toward selective residues and indicates that internalization and mains (39), whereas p75NTR binding is facilitated through four sorting of the receptor–ligand complex is blocked as a result of the negatively charged cysteine-rich repeats (19). It is likely that overall inhibition of the receptor tyrosine kinase activity by K252a. localization of the CTGF binding sites on these receptors will This is consistent with the hypothesis that TrkA activation is an ultimately lead to the generation of selective small molecule obligatory step in the recruitment to clathrin-coated pits and entry antagonists. 348 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 340–351, 2005

Figure 13. Inhibition of CTGF-dependent induction of TGF-␤– inducible early gene (TIEG) in HMC by K252a. Serum-starved HMC were preincubated with or without K252a (100 nM) for 30 Figure 11. NGF and CTGF activate TrkA in HMC. Serum- min and then stimulated with CTGF/V5 (40 ng/ml) in the starved HMC were incubated with CTGF/V5 (40 ng/ml) or presence or absence of K252a for 2 h, after which the cells were NGF (50 ng/ml) in the presence or absence of K252a (100 nM) lysed. Equal amounts of total lysate protein were subjected to for the period of time indicated, after which the cells were SDS-PAGE and analyzed by Western blotting using an anti- lysed. Equal amounts of lysate protein were subjected to SDS- TIEG antibody. Results are representative of three separate PAGE and analyzed by Western blotting using phospho-spe- experiments. cific antibodies against TrkA (Tyr674/675) or total TrkA. Re- sults are representative of three separate experiments. nephropathy (41,42). It was concluded that p75NTR is a marker of mesangial cell injury or activation and a potential molecule for signaling mechanisms to recruit or activate cells at sites of tubulointerstitial injury. NGF receptors were also detected on activated murine CD4 T cells (but not on resting T cells) (43), human B lymphocytes (44), monocytes (45), skin and lung fibroblasts (46), keratinocytes (40), melanocytes (47), periarte- rial connective tissue cells (40), and human osteoarthritic chondrocytes (48). The finding that CTGF acts via TrkA/p75NTR is intriguing, as there are some similarities between CTGF and neurotrophins. All of the neurotrophins are initially synthesized and secreted as 30- to 35-kD precursor proteins. These are proteolytically cleaved in the middle to release the biologically active 12- to 14-kD C-terminal mature forms (49). It is proposed that the N-terminal domain allows for correct protein folding and se- cretion (49,50), and it may also possess some biologic activities (51). However, secreted unprocessed immature or proneurotrophins are often present in abundance (52). CTGF is also synthesized and secreted as a 36- to 38-kD peptide, but different forms between 10 and 20 kD are also detected and are believed to be proteolytic products of the Figure 12. Dose-dependent effect of K252a on TrkA autophos- 38-kD form (53). The C-terminal 10- to 12-kD peptide is biolog- phorylation and signaling in HMC. Serum-starved HMC were ically active. pretreated or not with K252a at the concentrations indicated for Mature neurotrophins dimerize and adopt a novel tertiary 30 min and then were stimulated with 40 ng/ml CTGF/V5 for fold as a result of a cystine knot motif present in each monomer 15 min. (A) Cells were lysed, and 25 ␮g of total protein was (54). This cystine knot is present in the C-terminal of CTGF (55). subjected to SDS-PAGE for Western blot analysis. Membranes were probed with the primary antibodies indicated. (B through Moreover, CTGF contains a characteristic heparin-binding do- D) Densitometric quantification of blots. Results (mean Ϯ SEM) main in its C-terminus. Similarly, two novel NGF-like neuro- represent three separate experiments (n ϭ 6). *P Ͼ 0.0001. trophins, NT-6/7, that contain an additional 15 to 22 amino acid residues with heparin-binding ability in their mature forms have been identified (56,57). They were found to bind p75NTR It is of interest that NGF receptors have been detected in and activate TrkA but not TrkB or TrkC (56,57). many nonneuronal cells under normal and pathologic condi- It is interesting that it has been shown that mature neurotro- tions. Increased TrkA expression was reported in human skin phins dimerize and preferentially activate the Trk, whereas the during diabetes (40). Low expression of p75TNR was reported in unprocessed precursor forms preferentially activate p75NTR to normal glomeruli and in a subpopulation of renal interstitial induce a proapoptotic response (26,58). Thus, it has been pro- cells. However, the level of this receptor was found to be posed that the survival or death of neurones that coexpress the increased during inflammatory kidney diseases and in diabetic N-terminal domain and a Trk receptor could depend on pro- J Am Soc Nephrol 16: 340–351, 2005 CTGF Activates the TrkA Receptor 349 cessing of the neurotrophin ligands (59). Activation of p75NTR nective-tissue growth factor (CTGF) modulates cell signal- by proneurotrophins may also promote other functions ing by BMP and TGF-beta. Nat Cell Biol 4: 599–604, 2002 through pathways involving increased NF-␬B or c-Jun kinase 5. 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Correction dose of ARB (irbesartan 150 mg/day) and in 15% of the control Schmieder et al.: Additional Antiproteinuric Effect of Ultra- group who received placebo on top of other antihypertensive high Dose Candesartan: A Double-Blind, Randomized, Pro- medication (32). spective Study. J Am Soc Nephrol 16: 3038–3045, 2005. The authors regretfully report an error in quoting the find- ings of the Irbesartan in Patients with Type 2 Diabetes and Correction Microalbuminuria 2 (IRMA 2) Study at the bottom of the left Wahab et al.: Connective Tissue Growth Factor CCN2 Inter- column on page 3043. The second sentence of the paragraph acts with and Activates the Tyrosine Kinase Receptor TrkA. beginning, “Various clinical studies support. . . ,” should read J Am Soc Nephrol 16: 340–351, 2005. as follows: In this article, Figure 5 is in fact a duplicate of Figure 12. The largest trial in patients with type 2 diabetes and mi- Please see the correct Figure 5 below. The legend for Figure 5 is croalbuminuria, the Irbesartan in Patients with Type 2 Diabetes correct as published. The publisher regrets this error. and Microalbuminuria 2 (IRMA 2) Study, found that progres- Also in this article, the authors would like to correct a refer- sion of diabetic nephropathy occurred in approximately 5% of ence to Figure 13. On page 347, in the second line from the top patients who had diabetes and received a high dose of ARB of the right column, the textual reference to Figure 11 should (irbesartan 300 mg/day): In 10% of those who received a low refer instead to Figure 13. The authors regret this error.