A Novel Role for the Adaptor Molecule CD2-associated Protein in Transforming Growth Factor-β-induced Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m403534200
ISSN1083-351X
AutoresMario Schiffer, Peter Mündel, Andréy S. Shaw, Erwin P. Böttinger,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoCD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephrotic syndrome and renal failure caused by glomerulosclerosis. Here we report that increased transforming growth factor-β1 (TGF-β1) expression and apoptosis were present in podocytes at the onset of albuminuria and were followed by depletion of podocytes associated with progressive focal-segmental glomerulosclerosis in CD2AP-/- mice. Conditionally immortalized podocytes derived from CD2AP-/- mice were more susceptible to TGF-β-induced apoptosis compared with CD2AP+/+ podocytes. Reconstitution of CD2AP rescued CD2AP-/- podocytes from TGF-β-induced apoptosis. CD2AP was required for early activation of anti-apoptotic phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 1/2 by TGF-β. In contrast, activation of pro-apoptotic p38 MAPK by TGF-β was accelerated and enhanced in the absence of CD2AP. CD2AP was not required for PI3K/AKT activation by insulin and epidermal growth factor, indicating that CD2AP is a selective mediator of anti-apoptotic TGF-β signaling. In summary, we identified CD2AP as a novel mediator for selective activation of survival pathways and repression of apoptosis signaling by TGF-β in podocytes. Together, our in vitro and in vivo findings suggest that TGF-β-induced podocyte apoptosis is an early pathomechanism in mice developing focal-segmental glomerulosclerosis associated with functional impairment of CD2AP. CD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephrotic syndrome and renal failure caused by glomerulosclerosis. Here we report that increased transforming growth factor-β1 (TGF-β1) expression and apoptosis were present in podocytes at the onset of albuminuria and were followed by depletion of podocytes associated with progressive focal-segmental glomerulosclerosis in CD2AP-/- mice. Conditionally immortalized podocytes derived from CD2AP-/- mice were more susceptible to TGF-β-induced apoptosis compared with CD2AP+/+ podocytes. Reconstitution of CD2AP rescued CD2AP-/- podocytes from TGF-β-induced apoptosis. CD2AP was required for early activation of anti-apoptotic phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 1/2 by TGF-β. In contrast, activation of pro-apoptotic p38 MAPK by TGF-β was accelerated and enhanced in the absence of CD2AP. CD2AP was not required for PI3K/AKT activation by insulin and epidermal growth factor, indicating that CD2AP is a selective mediator of anti-apoptotic TGF-β signaling. In summary, we identified CD2AP as a novel mediator for selective activation of survival pathways and repression of apoptosis signaling by TGF-β in podocytes. Together, our in vitro and in vivo findings suggest that TGF-β-induced podocyte apoptosis is an early pathomechanism in mice developing focal-segmental glomerulosclerosis associated with functional impairment of CD2AP. The transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF, transforming growth factor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; CD2AP, CD2-associated protein; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; FSGS, focal-segmental glomerulosclerosis; CMS, Cas ligand with multiple SH3 domains. superfamily consists of secreted peptides, of which the three TGF-β isoforms (TGF-β1-3), activins, and bone morphogenetic proteins are best known in mammalian development, homeostasis, and pathobiology. The TGF-β isoforms are widely expressed and act on virtually every cell type in mammals by engaging a ubiquitous intracellular signaling cascade of Smad family proteins through ligand-induced activation of heteromeric transmembrane TGF-β receptor kinases. Receptor-activated Smad protein complexes accumulate in the nucleus where they participate directly in transcriptional activation of target genes. In addition, TGF-β receptors can activate Smad-independent signaling mechanisms, including mitogen-activated protein kinases (MAPKs), and PI3K (1Massague J. Nat. Rev. Mol. Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1658) Google Scholar, 2Roberts A.B. Cytokine Growth Factor Rev. 2002; 13: 3-5Crossref PubMed Scopus (74) Google Scholar). However, molecular mechanisms of activation of Smad-independent pathways by TGF-β receptors remain unclear. The mouse CD2 receptor-associated protein (CD2AP) and its human orthologue Cas ligand with multiple SH3 domains (CMS) belong to a family of ubiquitously expressed adaptor molecules that also includes the human Cbl-interacting protein of 85 kDa (CIN85) and its rat and mouse orthologue regulator of ubiquitous kinase (Ruk) and SH3 domain-containing gene expressed in tumorigenic astrocytes (SETA) (for review, see Ref. 3Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (165) Google Scholar). These proteins are defined as cytoplasmic adaptor or scaffolding proteins by three N-terminal SH3 domains, a proline-rich domain, and a C-terminal coiled-coil domain. CIN85/CMS family proteins have been shown to interact with proline-rich regions present in a variety of signaling proteins, including Cbl oncogene, CD2 receptor, AIP1 apoptosis-inducing protein-1, and the p85 subunit of phosphatidylinositol 3-kinase (PI3K), mediated through the SH3 domains, and with focal adhesion kinase p130Cas, Src family kinases Fyn, Src, and Yes, Grb2, and endophilins, mediated through the proline-rich region (for review, see Ref. 3Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (165) Google Scholar). Other structural features include FXDXF motifs, mediating interactions with endocytic proteins, and actin binding motifs, mediating interactions with actin cytoskeleton. Based on these observations, CIN85/CMS proteins are thought to exert multiple and diverse signaling functions in the organization of the immunological synapse in T cells, endocytosis and signaling of receptor tyrosine kinases, and neuronal apoptosis (3Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (165) Google Scholar). Degeneration of differentiated glomerular, tubular, and vascular cells in the normal nephron is a hallmark of chronic progressive kidney disease along with readily apparent renal scarring (4Rennke H.G. Anderson S. Brenner B.M. Tisher C.C. Brenner B.M. Renal Pathology. J. B. Lippincott Co, Philadelphia1994: 116-150Google Scholar, 5Bohle A. Wehrmann M. Bogenschutz O. Batz C. Vogl W. Schmitt H. Muller C.A. Muller G.A. Pathol. Res. Pract. 1992; 188: 908-924Crossref PubMed Scopus (122) Google Scholar, 6Fine L.G. Orphanides C. Norman J.T. Kidney Int. Suppl. 1998; 65: 74-78PubMed Google Scholar). Sclerosing glomeruli are characterized by progressive depletion of podocytes, resulting in denuded glomerular basement membrane areas and tuft adhesions, which may be considered as initial lesions of irreversible glomerular injury (4Rennke H.G. Anderson S. Brenner B.M. Tisher C.C. Brenner B.M. Renal Pathology. J. B. Lippincott Co, Philadelphia1994: 116-150Google Scholar, 7Kriz W. Gretz N. Lemley K.V. Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). Indeed, recent studies demonstrate that podocyte numbers are reduced early in glomeruli of diabetic patients with nephropathy (8Meyer T.W. Bennett P.H. Nelson R.G. Diabetologia. 1999; 42: 1341-1344Crossref PubMed Scopus (387) Google Scholar, 9Steffes M.W. Schmidt D. McCrery R. Basgen J.M. Kidney Int. 2001; 59: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). We have shown that TGF-β1 induces apoptosis in podocytes in vitro and in vivo via a p38 MAPK and caspase-dependent mechanism. We further demonstrated in a TGF-β1 transgenic mouse model of glomerulosclerosis that podocyte apoptosis is an early glomerular phenotype leading to progressive podocyte depletion. Together, our studies suggest that TGF-β may induce podocyte apoptosis and depletion in glomerulosclerosis (10Schiffer M. Bitzer M. Roberts I.S. Kopp J.B. ten Dijke P. Mundel P. Bottinger E.P. J. Clin. Investig. 2001; 108: 807-816Crossref PubMed Scopus (553) Google Scholar). An important functional role for CMS/CD2AP in the kidney was suggested initially by phenotype features in CD2AP knockout mice (CD2AP-/-), which manifest high grade albuminuria at a young age followed by progressive glomerulosclerosis and tubulointerstitial fibrosis and death from renal failure (11Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (700) Google Scholar). In the present study we describe a novel and essential role for CD2AP in TGF-β signaling in podocytes. We demonstrate that CD2AP is required for rapid activation of PI3K and ERK MAPK pathways by TGF-β. Failure of TGF-β receptors to engage these anti-apoptotic pathways early in the absence of CD2AP is associated with hyperactivation of proapoptotic p38 MAPK and accelerated apoptosis in podocytes in vitro. Finally, rates of apoptosis and expression of TGF-β1 were greatly increased, specifically in podocytes in the kidneys of CD2AP-/- mice, coincident with the onset of albuminuria, and this preceded podocyte depletion and glomerulosclerosis. Mice—CD2AP knockout mice in mixed C57Bl6/129J background were described previously (11Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (700) Google Scholar). Kidney tissue was harvested and either immersion-fixed in formaldehyde for paraffin embedding or embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) for frozen sections. Animal protocols and procedures were reviewed for ethical and humane standards and approved by an institutional Animal Use Committee. Cell Culture—Conditionally immortalized podocytes were derived from CD2AP-/- mice and CD2AP+/+ littermates and cultured as previously reported (12Mundel P. Kriz W. Exp. Nephrol. 1996; 4: 263-266PubMed Google Scholar). In brief, podocytes were grown on collagen type I (BD Biosciences) at 33 °C with interferon-γ (10 units/ml; Invitrogen) to drive expression of thermosensitive SV40 large T antigen. To induce differentiation, podocytes were maintained at 37 °C without interferon-γ for 14 days. Cytokine and Inhibitor Treatment—Proliferating CD2AP+/+ and CD2AP-/- podocytes were expanded to 70% confluence and serum-deprived by culturing in 0.2% fetal bovine serum for 24 h before stimulation with 5 ng/ml recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) in the absence or presence of 10 μm AKT inhibitor SH-5 (d-3-deoxy-2-O-methyl-myo-inositol 1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]) (13Kozikowski A.P. Sun H. Brognard J. Dennis P.A. J. Am. Chem. Soc. 2003; 125: 1144-1145Crossref PubMed Scopus (197) Google Scholar) (Alexis Biochemicals, San Diego, CA), wortmannin (100 nm) (Calbiochem), or epidermal growth factor (EGF) (20 ng/ml) and insulin (25 ng/ml) (Sigma). Immunostaining in Situ and in Vitro—For in situ detection of proteins we used the following antibodies: monoclonal anti-synaptopodin antibody (14Mundel P. Anat. Anz. 1998; 180: 391-392Crossref Scopus (7) Google Scholar), rabbit polyclonal anti-WT-1 antibody (Santa Cruz Biotechnology, St. Cruz, CA), and anti-active TGF-β1 antibody (LC 1-30-1) (a gift from Anita Roberts, National Cancer Institute). The samples were evaluated on a Bio-Rad MR600 confocal microscope, and images were captured using a Kanton Electronic Prog/Res/3012 digital video camera and digitally processed using Adobe Photoshop 5.0.2 (Adobe Systems, Inc., San Jose, CA). Scoring of Apoptotic Nuclei in Situ—To detect DNA fragmentation in situ, apoptotic nuclei were detected by a terminal dUTP nick-end labeling assay of kidney sections using peroxidase and counterstaining with hematoxylin and periodic acid-Schiff stain, as described earlier (10Schiffer M. Bitzer M. Roberts I.S. Kopp J.B. ten Dijke P. Mundel P. Bottinger E.P. J. Clin. Investig. 2001; 108: 807-816Crossref PubMed Scopus (553) Google Scholar). The terminal dUTP nick-end labeling assay was used according to the manufacturer's protocol (Intergen Co., Purchase, NY). Positive cells were counted as podocytes when residing on the outer aspect of periodic acid-Schiff-positive basement membrane. Cells residing in areas circumscribed by periodic acid-Schiff-positive basement membrane were counted as endocapillary/mesangial cells. Cells lining the inner surface of the Bowman's capsule were counted as parietal epithelial cells. Quantification of Apoptotic Nuclei in Vitro—Podocytes were seeded on glass coverslips and cultured at 33 °C. After 24 h of serum starvation in 0.2% fetal bovine serum, cells were pretreated with chemical inhibitors as indicated and treated with or without 5 ng/ml TGF-β for 24 or 48 h as indicated before fixation in 2% paraformaldehyde. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma), and coverslips were mounted on slides. Apoptotic nuclei were counted in 20 fields per sample by a person blinded to the experimental conditions. Numbers were normalized for the total cell number per high power field. For reconstitution of CD2AP, cells were cotransfected with pcDNA3.1-myc-CD2AP or empty control vector pcDNA3.1 together with pEGFP (Clontech Laboratories Inc., Palo Alto, CA) in a ratio of 5:1 using Effectene transfection reagent (Qiagen Inc., Valencia, CA). Transfection efficiency was verified after 24 h by examination of enhanced GFP expression. Cells were serum-starved for 12 h before stimulation with or without TGF-β1 as indicated. Cell Death Δ405 Enzyme-linked Immunosorbent Assay (ELISA)—Apoptotic cell death was also quantified in vitro using the Cell Death Δ405 ELISA (Roche Diagnostics) according to the manufacturer's protocol. In brief, 104 cells were lysed in the provided lysis buffer, and lysates were placed in streptavidin-coated microtiter plates. Anti-histone biotin and anti-DNA peroxidase-labeled antibodies were added. The anti-histone antibody binds to the histone components of the nucleosomes and captures the immunocomplex to streptavidin via its biotinylation. The anti-DNA peroxidase reacts with the DNA components of the nucleosomes. After incubation and washing, color was developed using the provided 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate solution to the wells. Absorbance was measured at 405 nm. Western Blotting—To detect levels of total protein and phosphorylated proteins, cells were lysed in radioimmune precipitation assay buffer containing protease inhibitors and phosphatase inhibitors as previously described (15Schiffer M. Schiffer L.E. Gupta A. Shaw A.S. Roberts I.S. Mundel P. Bottinger E.P. J. Am. Soc. Nephrol. 2002; 13: 2657-2666Crossref PubMed Scopus (78) Google Scholar). Lysates were subjected to 10% SDS-PAGE before transfer to polyvinylidene difluoride membranes (PerkinElmer Life Sciences). Western blotting was performed as previously described, and the following primary antibodies were used for antigen detection: mouse monoclonal anti-total AKT1 (Santa Cruz Biotechnology), rabbit polyclonal anti-S(P)475 AKT, rabbit anti-phospho-Smad2, mouse anti-phospho-Erk1/2, rabbit anti-total ERK1/2, rabbit anti-phospho-p38, and mouse anti-total p38 (all from Cell Signaling Inc., Beverly, MA). Quantitative Real-time PCR—Glomerular fractions were prepared by a sieving method to >95% purity from fresh mouse kidneys. Total RNA was prepared from glomerular fractions using RNeasy columns (Qiagen, Hilden, Germany) and reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen), and the cDNA was amplified using SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA) and specific primers for murine TGF-β1 (5′ primer, CCCTATATTTGGAGCCTGGA; 3′ primer, GTTGGTTGTAGAGGGCAAGG), TGF-β-RI (5′ primer, TCCAAACAGATGGCAGAGC; 3′ primer, TCCATTGGCATACCAGCAT), TGF-β-RII (5′ primer, CAGTGTGCTGAGAGACCGAG; 3′ primer, AGCACTCGGTCAAAGTCTCA), murine WT-1 (5′ primer, ATCTGAAGACCCACACCAGG; 3′ primer, GCTGAAGGGCTTTTCACTTG), and murine Smad7 (5′ primer, ATCTTCATCAAGTCCGCCAC; 3′ primer, AACCAGGGAACACTTTGTGC) in an iCycler (Bio-Rad). Normalization across samples was performed using the average of the constitutive gene murine β-actin (5′ primer, ACCGTGAAAAGATGACCCAG; 3′ primer, AGCCTGGATGGCTACGTACA). Increased Expression of TGF-β1 in Podocytes in CD2AP-/- Mice Is Associated with Onset of Albuminuria and FSGS—We previously reported that Smad7 and TGF-β1 induce apoptosis in podocytes in vitro and in vivo (10Schiffer M. Bitzer M. Roberts I.S. Kopp J.B. ten Dijke P. Mundel P. Bottinger E.P. J. Clin. Investig. 2001; 108: 807-816Crossref PubMed Scopus (553) Google Scholar). To determine whether manifestations of FSGS typically observed in CD2AP-/- mice were associated with TGF-β expression profiles in glomeruli, we examined functional and histopathological manifestations of FSGS in CD2AP-/- and CD2AP+/+ littermates at 1, 2, 3, and 4 weeks of age. Immunoperoxidase labeling of kidney sections from 2-, 3-, and 4-week-old mice for TGF-β1 protein demonstrated that podocytes in 3-week-old (n = 5) (Fig. 1d) and 4-week-old CD2AP-/- mice (n = 5) (Fig. 1f) stained strongly positive for TGF-β1, whereas 3- and 4-week-old CD2AP+/+ controls (Fig. 1, c and e) and 2-week-old mice of either genotype showed no or minimal TGF-β1 staining in podocytes (Fig. 1, a and b). The strong increase in TGF-β1 protein synthesis in podocytes in this model is similar to the increase in Smad7 protein in podocytes that we observed previously in CD2AP-/- mice (15Schiffer M. Schiffer L.E. Gupta A. Shaw A.S. Roberts I.S. Mundel P. Bottinger E.P. J. Am. Soc. Nephrol. 2002; 13: 2657-2666Crossref PubMed Scopus (78) Google Scholar). During the first 2 weeks, kidney structure and function were not significantly different in CD2AP-/- and control littermates, respectively. However all CD2AP-/- animals had developed high grade albuminuria when examined at 3 weeks of age; significant glomerular histopathology characterized by mesangial proliferation and early FSGS lesions was detectable at 3 weeks and progressed to sclerosis in more than 50% of examined glomeruli in CD2AP-/- mice at 4 weeks (data not shown). Thus, the onset of glomerular disease in this model with high grade proteinuria is rapid and occurs between 2 and 3 weeks of age. Next, we isolated glomeruli from CD2AP+/+ and CD2AP-/- kidneys at 4 weeks of age to quantitate mRNA expression of key components of TGF-β signaling directly in primary glomerular fractions by quantitative real-time PCR. Transcript levels for TGF-β1, type II TGF-β receptor (TGF-βRII), and Smad7 were all significantly increased in glomeruli obtained from CD2AP-/- compared with glomeruli from CD2AP+/+ mouse kidneys (Fig. 1g). In contrast, type I TGF-β receptor mRNA was not significantly different (Fig. 1g). Together, these in situ protein and mRNA expression data indicate that increased expression of TGF-β1 and Smad7 by differentiated podocytes in CD2AP-/- mice is associated with albuminuria and mesangial activation at early stages of glomerulosclerosis. CD2AP Deficiency Predisposes Podocytes to Undergo Apoptosis Induced by Autocrine and Paracrine TGF-β in Vitro—After we determined that increased expression of mediators of TGF-β signaling in podocytes of CD2AP-/- mice coincided with onset of albuminuria, we wished to examine whether TGF-β signaling and apoptosis were affected by CD2AP deficiency in vitro. Conditionally immortalized CD2AP-/- and control CD2AP+/+ podocyte cell lines were established from littermate mice as described previously (12Mundel P. Kriz W. Exp. Nephrol. 1996; 4: 263-266PubMed Google Scholar). After 14 days of culture in non-permissive conditions, morphology and expression patterns of podocyte markers such as WT-1, synaptopodin, and podocin was indistinguishable in CD2AP-/- and control CD2AP+/+ podocyte cell lines (data not shown). In addition, distribution patterns of ZO-1 protein indicated the formation of characteristically interdigitated cell extensions in cultured CD2AP-/- and CD2AP+/+ podocytes (data not shown). Three independent cell clones for each genotype were examined. Quantitation of apoptotic nuclei showed that base-line (un-treated) rates of apoptosis were significantly higher in CD2AP-/- compared with CD2AP+/+ (2.4 ± 0.9 versus 0.7 ± 0.3%, respectively) (Fig. 2a). TGF-β treatment for 24 h increased apoptosis rates by 2.5 ± 0.5-fold over base line in CD2AP+/+ cells and 4.8 ± 1.7-fold over base line in CD2AP-/- podocytes (Fig. 2a). Thus, the apoptosis-inducing effect of TGF-β was significantly enhanced in CD2AP-/- compared with CD2AP+/+ podocytes (4.8 ± 1.7 versus 2.5 ± 0.5-fold induction; p < 0.03). These findings suggest that CD2AP exerted an anti-apoptotic function in podocytes both at base line and in particular when exposed to exogenous (paracrine) TGF-β1. Because we have found that podocytes also secrete latent and active autocrine TGF-β2, 2D. Wu and E. P. Böttinger, unpublished observations. we examined base-line apoptosis rates in the absence or presence of pan-neutralizing anti-TGF-β antibody 2G7. Neutralizing antibody (2G7) significantly reduced base-line apoptosis in CD2AP-/- podocytes by 49% compared with control IgG (1.09 ± 0.39 versus 2.13 ± 0.22%, p < 0.004) (Fig. 2b). Thus, increased base-line apoptosis observed in CD2AP-/- podocytes is largely, but not exclusively, dependent on autocrine TGF-β signaling, indicating that TGF-β receptor activity and survival pathway signaling are coupled in podocytes by the CD2AP adaptor molecule. We conclude that CD2AP deficiency predisposes podocytes to apoptosis induced by both exogenous (paracrine) and autocrine TGF-β. CD2AP Is Required for Rapid Activation of PI3K and ERK1/2 by TGF-β but Not by EGF and Insulin—We reasoned that the anti-apoptotic effect of CD2AP on TGF-β-induced apoptosis could be due to essential survival signaling and/or inhibition of pro-apoptotic pathways or both. Because PI3K/AKT functions as a primary cell survival pathway in many cell types (16Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar), we examined activation profiles of PI3K/AKT in response to TGF-β1 in three independent lines of CD2AP-/- and CD2AP+/+ podocytes, respectively. In CD2AP+/+ podocyte lines, TGF-β1 induced a strong and rapid (minutes) phosphorylation of AKT on serine 473 (Fig. 3a). Serine 473 phosphorylation peaked between 1 and 2 h and remained elevated for up to 4 h (Fig. 3b). In contrast, S(P)473-AKT was not detectable until 1 h of TGF-β treatment in CD2AP-/- podocyte lines (Fig. 3a), and peak phosphorylation was delayed at 4 h of TGF-β1 treatment (Fig. 3b). Among MAPKs, the ERK1/2 mediates primarily mitogenic and/or anti-apoptotic signaling (17Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3542) Google Scholar, 18Cross T.G. Scheel-Toellner D. Henriquez N.V. Deacon E. Salmon M. Lord J.M. Exp. Cell Res. 2000; 256: 34-41Crossref PubMed Scopus (620) Google Scholar). Interestingly, the profile of ERK1/2 activation in control CD2AP+/+ podocytes treated with TGF-β1 was very similar to the S(P)473-AKT profile, including a strong and rapid activation detectable at 5 min (Fig. 3a) followed by peak phosphorylation at 2 h (Fig. 3b). Similar to S(P)473-AKT, phosphorylation of ERK1/2 was not detectable during the initial 45 min (Fig. 3a), and peak phosphorylation was delayed between 2 and 4 h in CD2AP-/- podocytes (Fig. 3b). Thus, the anti-apoptotic mediators PI3K/AKT and ERK1/2 are activated by TGF-β in podocytes with similar kinetic profiles, consistent with previous reports that ERK1/2 and PI3K are synergistically activated to mediate anti-apoptotic pathways (19Davies C.C. Mason J. Wakelam M.J. Young L.S. Eliopoulos A.G. J. Biol. Chem. 2004; 279: 1010-1019Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20Xu D. Kyriakis J.M. J. Biol. Chem. 2003; 278: 39349-39355Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Our results presented here demonstrate for the first time that early activation of PI3K/AKT and ERK1/2 MAPK by TGF-β in podocytes requires the CD2AP adaptor protein. To examine whether CD2AP and PI3K function in a linear pathway, we quantitated and directly compared the extent of TGF-β-stimulated apoptosis as assayed by DAPI count and cell death ELISA (Δ405) in CD2AP-/- podocytes and CD2AP+/+ podocytes pretreated with wortmannin, a chemical inhibitor of PI3K function. Pretreatment with wortmannin resulted in a significant increase of the proapoptotic TGF-β effect in CD2AP+/+ podocytes, but not in CD2AP-/- podocytes, as determined by DAPI (Fig. 4a) and cell death assay (Fig. 4b). Similar results were obtained when the AKT-specific inhibitor SH-5 was used in place of wortmannin (data not shown). These findings demonstrate that both CD2AP-deficiency or functional inactivation of PI3K were associated with enhanced TGF-β-induced apoptosis signaling in a non-additive, quantitatively comparable manner, indicating that CD2AP and PI3K function in a linear pathway downstream of TGF-β receptors. To confirm this hypothesis at a molecular level by direct reconstitution experiment, we transfected CD2AP-/- podocytes with CD2AP expression plasmid. Expression of epitope-tagged, reconstituted CD2AP in transfected CD2AP-/- cells was validated by Western blot analysis (data not shown). Rates of apoptotic nuclei were significantly reduced in CD2AP-/- cells transfected with CD2AP expression plasmid compared with empty vector (control)-transfected cells (Fig. 4c). Together these findings demonstrate that PI3K/AKT mediate survival signals in podocytes exposed to TGF-β1 and show that CD2AP is required for activation of this survival pathway by TGF-β. To examine whether CD2AP is a general or pathway-selective mediator of PI3K/AKT activation in podocytes, we treated CD2AP-/- and control CD2AP+/+ podocytes with insulin and EGF, two well characterized extracellular stimulators of PI3K/AKT-dependent anti-apoptotic signaling (21Bevan P. J. Cell Sci. 2001; 114: 1429-1430Crossref PubMed Google Scholar, 22Danielsen A.J. Maihle N.J. Growth Factors. 2002; 20: 1-15Crossref PubMed Scopus (91) Google Scholar). In contrast with TGF-β stimulation, insulin and EGF induced early AKT phosphorylation rapidly and to a similar extent in both CD2AP+/+ and CD2AP-/- podocytes, respectively (Fig. 4d), suggesting that CD2AP is required for activation of PI3K/AKT pathway by TGF-β signaling through receptor serine/threonine kinases but not by insulin or EGF signaling through receptor tyrosine kinases. We reasoned next that if CD2AP is not required for PI3K/AKT pathway activation by insulin and EGF, then pretreatment with insulin should reduce the increased apoptotic response induced by TGF-β irrespective of the absence or presence of CD2AP. Indeed, pretreatment with insulin significantly inhibited TGF-β-induced apoptosis in both control CD2AP+/+ and CD2AP-/- cells, providing further functional evidence that CD2AP is selectively required for PI3K/AKT-associated anti-apoptotic signaling by TGF-β but not by insulin (Fig. 4e). CD2AP Deficiency Is Associated with Hyperactivation of Proapoptotic p38 MAPK by TGF-β—We have previously shown that TGF-β activates p38 MAPK in podocytes and that p38 is required for TGF-β-induced podocyte apoptosis (10Schiffer M. Bitzer M. Roberts I.S. Kopp J.B. ten Dijke P. Mundel P. Bottinger E.P. J. Clin. Investig. 2001; 108: 807-816Crossref PubMed Scopus (553) Google Scholar). Base-line levels of phospho-p38 were increased in CD2AP-/- compared with control CD2AP+/+ podocytes (Fig. 5a). TGF-β-induced phosphorylation of p38 was detectable at 30 min in control CD2AP+/+ cells (Fig. 5a) and peaked at 2 h (Fig. 5b). In CD2AP-/- podocytes, TGF-β-induced phospho-p38 activation peaked earlier (1 h) and was further increased at 4 h when compared with CD2AP+/+ (Fig. 5b). The absence or presence of CD2AP had no effect on Smad2 phosphorylation by TGF-β receptors (Fig. 5, a and b), indicating that direct CD2AP-dependent signaling has no effect on Smad activation. Indeed, phospho-Smad2 profiles were comparable for up to 4 h in CD2AP+/+ and CD2AP-/- podocytes (Fig. 5b). Taken together, the kinetic profiles of PI3K/AKT and MAPK activation by TGF-β in podocytes suggest that ligand-activated TGF-β receptors control anti-apoptotic PI3K/AKT and ERK1/2 activation possibly through common upstream mediators, which include CD2AP as a novel essential signaling component between TGF-β receptor and PI3K. Moreover, TGF-β receptors control pro-apoptotic p38 activation through a pathway that is distinct from PI3K/AKT and ERK1/2. CD2AP-dependent signals negatively trans-modulate pro-apoptotic MAPKs. Thus, the net effect of loss of CD2AP function in TGF-β signaling is enhanced proapoptotic p38 signaling activity and diminished anti-apoptotic PI3K/AKT and ERK1/2 pathway activity, consistent with our finding of increased sensitivity to TGF-β1-induced apoptosis in CD2AP-/- podocytes. Podocyte Apoptosis Is an Initial Manifestation in Albuminuric CD2AP-/- Mice Developing Podocyte Depletion and Glomerulosclerosis—Because CD2AP deficiency in podocytes in vitro caused increased susceptibility to TGF-β-induced apoptosis, we examined whether apoptosis was increased in glomerular cells in CD2AP-/- mice in vivo. Glomerular cell turnover was examined by terminal dUTP nick-end labeling immunoperoxidase staining in combination with periodic acid-Schiff staining to demarcate glomerular basement membrane. We observed significantly increased apoptosis in 3-week-old CD2AP-/- mice compared with age-matc
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