Article |
Address correspondence to Cheryl B. Knudson, Dept. of Biochemistry, Rush Medical College, Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Tel.: (312) 942-8249. Fax: (312) 942-3053. email: cheryl_knudson{at}rush.edu
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Abstract |
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Key Words: CD44; bone morphogenetic protein; hyaluronan; chondrocyte; Smad1
Abbreviations used in this paper: BMP, bone morphogenetic protein; SBE, Smad-binding element.
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Introduction |
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Signaling by BMPs is initiated by their binding to serine/threonine kinase type II and type I receptors and subsequent activation of receptor-activated (R-) Smad proteins (Moustakas et al., 2001). The signaling pathway of BMP-7 (also known as osteogenic protein-1) consists of a principal type II receptor (ActR-II) that recruits and transphosphorylates the type I receptor ALK2 (Macias-Silva et al., 1998). The ALK2 receptor signals through the R-Smad Smad1. Phosphorylation of Smad1 leads to its dissociation from the type I receptor and the subsequent oligomerization of Smad1 with the comediator Smad4 for nuclear translocation. This signaling pathway is similar to that of BMP-2 and BMP-4 whereby after binding to BMPR-II, the type I receptor ALK3 or ALK6 activate the Smad1 pathway. TGFßR-I and TGFßR-II transduce TGF-ß responses through Smad2 and Smad3, two related R-Smads (Moustakas et al., 2001).
Exogenous BMP-7 induces an anabolic response in a variety of connective tissues, including cartilage. BMP-7 stimulates synthesis of aggrecan (the chief proteoglycan of cartilage) and type II collagen (Flechtenmacher et al., 1996) as well as CD44 and hyaluronan synthase-2 (Nishida et al., 2000a,b) in bovine and human articular chondrocytes. Endogenous BMP-7 expression is highest in the cell clusters in osteoarthritic cartilage (Chubinskaya et al., 2000)areas that may represent attempted repair. Thus, BMP-7 may regulate cartilage development as well as articular cartilage repair and homeostasis not only by its stimulation of the synthesis of the major cartilage ECM components, but also with two molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.
As with other tissues, cellmatrix interactions mediated via transmembrane receptors are responsible for maintaining cartilage homeostasis (Knudson and Knudson, 1993; Knudson, 2003). Articular chondrocytes express integrin as well as nonintegrin ECM receptors (Knudson and Loeser, 2002). These receptors, through interactions with their principal ligands, provide chondrocytes the means to "sense" changes in the ECM environmentchanges that may elicit a reparative response, matrix remodeling, or alternatively, cellular quiescence. The CD44 receptor serves as the primary receptor for the ECM macromolecule hyaluronan (Underhill, 1992), mediating both cellcell and cellmatrix interactions. The CD44H ("hematopoetic") isoform is a highly glycosylated type I receptor with a 72-aa cytoplasmic domain encoded by exon 20 (Screaton et al., 1992). The mRNA for CD44E ("epithelial") contains three additional exons (variants 8, 9, and 10) encoding additional protein motifs in the extracellular domain and the same exon 20 encoding the cytoplasmic domain. CD44H is expressed by chondrocytes. In limb development, modulation of hyaluronancell interactions during chondrogenic condensation parallels the initiation of CD44 expression (Knudson and Toole, 1987; Rousche and Knudson, 2002). In adult articular chondrocytes, CD44 serves as the critical link to retain hyaluronanproteoglycan aggregates to the chondrocyte cell surface (Chow et al., 1995). However, new evidence indicates that CD44 participates in the transduction of matrix cues.
In this work, a yeast two-hybrid screen was used to identify proteins that could potentially mediate CD44 signaling. The screen detected binding between the Smad1 protein and a full-length cytoplasmic domain construct of CD44. CD44Smad1 receptor interactions were confirmed by immunoprecipitations of cell lysates. CD44 receptors truncated in the COOH-terminal cytoplasmic domain (Fig. 1 A) did not have the capacity to interact with Smad1. Chondrocytes overexpressing truncated CD44 in the background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation, indicative of a dominant-negative function. Additional evidence is presented to support that hyaluronanCD44 binding augments the cellular response to BMP-7, including Smad1 phosphorylation and induced nuclear translocation of Smad1 and Smad4. The chondrocyte response to matrix disruption or damage is likely mediated by cross-talk between receptors to ECM components and BMPs (Reddi, 1998). Thus, our data provide further support for the emerging paradigm that cellmatrix interactions modulate the cellular response to morphogens or growth factors.
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Results |
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Expression of CD44 constructs transfected into COS-7 cells and chondrocytes
Two model systems were used in this paper, COS-7 cells that do not express CD44 and bovine articular chondrocytescells that express CD44 and exhibit an extensive hyaluronan-dependent matrix. The anti-CD44 mAb BU-52 recognizes an extracellular epitope present in human CD44H and CD44E, as well as the COOH-terminal truncation mutants CD44H54 and CD44H
67 (Fig. 1 A). As shown in Fig. 1 B, nontransfected COS-7 cells (visualized as blue DAPI-stained nuclei) do not exhibit cell surface immunostaining for CD44. Upon transfection with full-length human pCD44H (successfully transfected cells are GFP positive), the COS-7 transfectants now displayed prominent cell surface immunostaining for CD44 with BU-52 (Fig. 1 B, red fluorescence). Transfection of COS-7 cells with other isoforms, pCD44E (Fig. 1 C) and the truncation mutant pCD44H
67 (Fig. 1 E), followed by BU-52 staining demonstrated that recombinant receptors were successfully processed and transported to the plasma membrane. The pCD44H/V5 construct, containing a COOH-terminal V5-epitope tag, could be detected after cell permeabilization using an anti-V5 mAb (Fig. 1 D). By Western blot analysis of total cell lysates (Fig. 1 F) we confirmed that nontransfected COS-7 cells (lane 1) do not express CD44, as reported in our previous paper (Jiang et al., 2002). Lysates from COS-7 cells transfected with pCD44H exhibited an immunoreactive band at
85 kD (lane 2), which is the signature molecular mass of full-length, highly glycosylated CD44H. Lysates from COS-7 cells transfected with the larger pCD44E construct exhibited, as expected, a fairly broad band at
130 kD (lane 5). Lysates from cells transfected with the COOH-terminal truncation mutants pCD44H
67 (lane 3) or pCD44H
54 (lane 4) also exhibited protein bands of the expected size range. Thus, in the COS-7 model the expression of CD44 can be selectively and specifically modified.
Primary cultures of bovine chondrocytes represent a more biologically relevant model system. In these cells, transfection of a COOH-terminal truncation mutant pCD44H67 is used as a dominant-negative to suppress the activity of the endogenous full-length CD44 (Jiang et al., 2002). Successfully transfected bovine chondrocytes can be visualized readily as GFP-positive cells. In addition, processing and transport of the human CD44H
67 to the plasma membrane in these cells can be visualized by BU-52 immunostaining (Fig. 1 G, red fluorescence). Bovine chondrocytes express abundant CD44, but are not recognized by antihuman CD44 antibodies such as BU-52 (Fig. 1 H; Aguiar et al., 1999).
Coimmunoprecipitation of CD44 and Smad1
The interaction between CD44 and Smad1 was confirmed by coimmunoprecipitation assays. COS-7 cells were cotransfected with myc-tagged pSmad1 together with pCD44H or pCD44H67. The cells were lysed and proteins were immunoprecipitated using an anti-myc antibody followed by Western blotting with anti-CD44 or anti-Smad mAb. Immunoprecipitation of Smad1 resulted in the coprecipitation of CD44H (Fig. 2 A, lane 1). The COOH-terminal truncation mutant CD44H
67 was used as a control and displayed no coprecipitation with Smad1 (Fig. 2 A, lane 2). To confirm that CD44H and CD44H
67 were present in the two cell lysates, the immunoprecipitation supernatants were analyzed. As shown in Fig. 2 A, nearly equivalent levels of CD44H (lane 4) and CD44H
67 (lane 5) were present in the original cell lysates. To address whether both lysates contained equal concentrations of Smad1, the same immunoprecipitate blot (Fig. 2 A) was reprobed with an anti-Smad1 antibody. As shown in Fig. 2 B, lanes 1 and 2, the anti-myc antibody immunoprecipitated nearly equal levels of myc-tagged Smad1, which can be seen as bands of
55 kD. Thus, immunoprecipitation of myc-Smad1 resulted in the coprecipitation of CD44H. However, under the same conditions there was no coprecipitation of the CD44 isoform CD44H
67 in which most of the cytoplasmic domain has been eliminated.
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To confirm the interaction of CD44 with endogenous Smad1, COS-7 cells were transfected with pCD44 isoforms containing a V5-epitope tag. COS-7 cells expressing CD44H/V5, CD44E/V5, or empty vector (pTracer-V5) were lysed and immunoprecipitated using an anti-V5 mAb. The immunoprecipitates were split into two aliquots and were resolved by SDS-PAGE followed by electroblotting. In Fig. 3, the top panel depicts the detection of Smad1 with anti-Smad1 antibody and the bottom panel the detection of CD44 with an anti-CD44 pAb. Lane 1 displays recombinant Smad1 as a molecular weight marker (top blot) and as molecular weight markers in the bottom blot. As shown in lane 2, COS-7 cells transfected with empty vector displayed no coprecipitation of endogenous Smad1. However, the cells transfected with either CD44H (lane 3) and the larger CD44E (lane 4) both displayed coprecipitation of endogenous Smad1. The CD44E contains an identical intracellular domain as CD44H, but has a longer extracellular domain sequence. To confirm the immunoprecipitations, the precipitated lysates were probed on a separate blot using an anti-CD44 antibody. From control COS-7 cells (Fig. 3, lane 2, bottom), only a nonspecific band at 124 kD was observed. Lysates from COS-7 cells transfected with CD44H exhibited an 85-kD band as well as the nonspecific band (lane 3, bottom), and CD44E-transfected cells, a wide band at
130 kD (lane 4, bottom). These CD44Smad1 coimmunoprecipitations confirm that endogenous Smad1 present in a mammalian cell type can form interactions with two of the common CD44 isoforms, CD44H and CD44E.
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Discussion |
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Our observations are consistent with a role for CD44-Smad1 binding in the augmentation of the cellular response to BMPs. CD44 could anchor Smad1 in resting cells for presentation to the type I BMP receptor. After BMP stimulation, we observed a reduction in CD44-Smad1 coimmunoprecipitation as nuclear translocation of Smad1 and Smad4 proceeded. As a precedent, other cytosolic molecules that control the access of R-Smads to the activated type I receptors have been described. Smad anchor for receptor activation (SARA) presents Smad2 or Smad3 to the activated TGFß receptor complex. SARA binds to the MH2 domain of Smad2, inducing a conformational change to increase the efficiency of receptor-mediated Smad2 phosphorylation (Wu et al., 2000). The phosphorylation releases Smad2/3 from SARA, allowing Smad4 binding and translocation to the nucleus. As well, the actin-binding protein filamin was found to be a Smad2-binding protein. Filamin-deficient melanoma cells were defective in TGFß signaling, but transient expression of filamin restored the response of TGFß-reporter gene activation and Smad2 nuclear accumulation (Sasaki et al., 2001). These investigators proposed that filamin might serve as an anchor protein analogous to SARA, to maintain localization of Smad2 near TGFß receptors or to present Smad2 in a conformation to facilitate its phosphorylation. CD44 may function in an analogous manner. Gain of CD44 expression in the COS-7 transfectants resulted in both the more unambiguous cytoplasmic localization of Smad1 in the resting transfectants and nuclear translocation of endogenous Smad1 in response to BMP-7 stimulation.
Our current analyses provide additional insight into the mechanism of Smad1 presentation to its cognate receptor kinase as described in other models (Moustakas et al., 2001; Qin et al., 2001). Our data suggest that the Smad1-binding site in the CD44 cytoplasmic domain is distal to the point of the CD4454 truncation. Phosphorylation of serine residues in the cytoplasmic domain of CD44 can modulate receptor function (Lewis et al., 2001). The forkhead associated (FHA) domain of Smad1 is a phosphopeptide-binding domain common to many proteins. Smad1-CD44 binding, potentially via a FHAphosphoserine interaction, could also participate in the recruitment and/or presentation of Smad1 to its type I receptor. Enzymatic removal of hyaluronan reduced CD44 phosphorylation (unpublished data), which might diminish Smad1-CD44 binding.
Notch and CD44 are receptors without catalytic activity in the cytoplasmic domain, but which undergo regulated intramembrane proteolysis (Cichy and Pure, 2003; Thorne et al., 2004). An extracellular domain cleavage followed by -secretase cleavage of the transmembrane domain releases the Notch intracellular domain (ICD; De Strooper et al., 1999) or the CD44 ICD (Lammich et al., 2002; Murakami et al., 2003), which exhibit nuclear translocation. Our results imply the capacity for Smad1 binding to the CD44 ICD. The CD44 ICD can cooperate with the p300/CREB-binding protein (CBP) to activate transcription (Okamoto et al., 2001). Smad1 also acts with p300/CBP to regulate transcriptional activation by BMPs (Pouponnot et al., 1998). In light of these previous reports, our findings suggest that Smad1 together with the CD44 ICD may function as accessory modulators of transcriptional regulation.
The shedding of CD44 after MT1-MMP cleavage of the extracellular domain has been proposed as a mechanism to regulate cell detachment from hyaluronan (Kajita et al., 2001). In addition to proteolytic cleavage of CD44, the receptor is internalized (Aguiar et al., 1999) as part of uptake of hyaluronan to the lysosomal compartment (Knudson and Knudson, 1993). The half-life of cell surface CD44 also decreases in the absence of its ligand, hyaluronan (Aguiar et al., 1999). Together with reduction in CD44 phosphorylation in the absence of ligand or increased expression of CD44exon19 as described above, all these are possible mechanisms to limit the participation of CD44 in the BMP signaling pathway.
Our results indicate that the regulation of functional CD44 and CD44 receptor ligation by hyaluronan influences the physiological functions for CD44Smad1 interaction to modulate the cellular response to BMP-7. Thus, it is possible that changes in CD44hyaluronan interactions during embryogenesis may regulate the cellular response to BMP. Stable CD44hyaluronan binding maintains cartilage homeostasis. Cartilage matrix disruption by interleukin-1 (Aydelotte et al., 1992) or fragments of the ECM (Chow et al., 1995; Knudson et al., 2000) results in an imbalance between ECM degradation and anabolic responses by chondrocytes. The intersection of CD44 with the BMP signal transduction pathways may explain this dysregulation. The evidence that Smad1 interacts with the cytoplasmic domain of CD44 supports an emerging paradigm wherein changes in the ECM can modulate BMP signal transduction, culminating in the influence on cellular metabolism.
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Materials and methods |
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Bovine articular chondrocytes were isolated (Chow et al., 1995), cultured for a short term in alginate beads (Nishida et al., 2000b), and then released and plated at 9 x 106 cells/100-mm dish as monolayer cultures in DMEM + 10% FBS overnight. For transfection, the chondrocytes were pretreated with 5 U/ml Streptomyces hyaluronidase (Sigma-Aldrich) in serum-free DMEM. LipofectAMINE 2000 was incubated with pCD44H, pCD44H67, or pCD44H
54, or with the pTracer-SV40 empty vector (facilitator/DNA ratio, 2.5:1), and lipid/DNA complexes were added at 15 µg DNA/107 cells (Jiang et al., 2002). FBS was added 5 h after transfection to a final concentration of 20%. 24 h after transfection, the medium was replaced with DMEM + 10% FBS and analysis began after 48 h. Transfected bovine chondrocytes express GFP and the human CD44H (or CD44H
67 or CD44H
54) protein at the cell surface as detected with BU-52 mAb.
Yeast two-hybrid screen
Two yeast two-hybrid baits were constructed by subcloning cDNAs encoding aa 292361 (full-length CD44 cytoplasmic domain) and aa 292307 (CD4454 COOH-terminal truncation) of the human CD44 receptor into the DNA-binding domain fusion vector pMW101 (Watson et al., 1996). The yeast two-hybrid screening protocol was performed as described previously (Slentz-Kesler et al., 2000) using cDNA libraries derived from human prostate and human macrophages.
Coimmunoprecipitation assays and Western blot analyses
48 h after transfection, the cells were lysed in 20 mM Tris, pH 7.4, with 50 mM NaCl, 5 mM EDTA, 3 mM MgCl2, 0.5% Triton-X-100 buffer, with protease (P2714, Sigma-Aldrich) and phosphatase inhibitors (P2850, Sigma-Aldrich). The cell lysates were precleared by protein GSepharose (Zymed Laboratories) followed by an overnight incubation at 4°C with anti-myc antibody (Invitrogen) and protein GSepharose beads. The beads were washed twice with lysis buffer and the immunoprecipitates were eluted using 100 mM glycine solution, pH 2.6, boiled for 10 min under reducing conditions before loading for SDS-PAGE using a 10% Tris-HCl gel. PVDF membrane was used in the electrotransfer. 5% milk in PBS-Tween was used as a blocking solution. The blot was first probed for CD44 using biotinylated BU-52 antibody (ID Labs), detected with Streptavidin-HRP and ECL reagents (Amersham Biosciences). After 4 d soaking in water, the same blot was next probed for Smad1 after the initial HRP-Streptavidin signal had faded. The anti-Smad-1 pAb (Upstate Biotechnology) was detected using a Vectastain kit (Vector Labs), and incubation with HRP-Streptavidin was followed by ECL reagents. Alternatively, COS-7 transfectants were lysed in 50 mM Tris, pH 7.4, with 150 mM NaCl, 3 mM MgCl2, and 0.5% NP-40 buffer, and with protease and phosphatase inhibitors. Pre-cleared lysates were incubated with the anti-V5 mAb and protein GSepharose overnight. The immunoprecipitates were analyzed by SDS-PAGE on a 7.5% gel followed by Western blotting. CD44 was detected with an anti-CD44 pAb (H-300; Santa Cruz Biotechnology, Inc.) and endogenous Smad1 was identified using the anti-Smad1 antibody.
Bovine chondrocytes were treated for 60 min with 100 ng/ml BMP-7 (R&D Systems) in the absence or presence of Streptomyces hyaluronidase (5 U/ml, 90 min pretreatment followed by a second addition of enzyme at the time of addition of BMP-7), or were left untreated. Total cell lysates were prepared (with the 50 mM Tris buffer above) and analyzed by SDS-PAGE on a 10% gel followed by Western blotting. Total Smad1 and phosphorylated Smad1 were detected with anti-Smad1 and anti-phospho-Smad1 pAbs, respectively (Upstate Biotechnology; Macias-Silva et al., 1998). Actin was detected by a ß-actin peptide mAb (AC-15; Sigma-Aldrich). The resultant band intensities were quantified using a Fluor-S imaging system (Bio-Rad Laboratories).
Immunostaining and Smad nuclear translocation
COS-7 stable transfectants (CD44+), control chondrocytes, or chondrocytes after transfection were incubated overnight in media containing only 0.5% FBS. Cells were lifted with nonenzymatic cell dissociation solution (Sigma-Aldrich), incubated for 60 min with 100 ng/ml BMP-7, and then fixed with 2% PFA, permeabilized with 0.2% Triton X-100, and incubated with an anti-Smad1 or an anti-Smad4 antibody (Upstate Biotechnology), which were detected using rhodamine red-X goat antirabbit IgG (Jackson ImmunoResearch Laboratories). Some chondrocytes were pretreated with Streptomyces hyaluronidase. All cells were incubated with DAPI (Molecular Probes, Inc.) as a nuclear counterstain. The cells were viewed using a microscope (Ellipse E600; Nikon) equipped with Y-Fl Epi-fluorescence, PLAN-Apochromat 1.4 NA/60x oil and PLAN 0.5 NA/20x objectives. Images were captured digitally in real time using a camera (Spot-RT; Diagnostic Instruments) and were processed using MetaView imaging software (Universal Imaging Corp.).
Protein/DNA array analysis of transcription factor activation
Nuclear extracts isolated from bovine articular chondrocytes were treated for 60 min with 100 ng/ml BMP-7, in the presence or absence Streptomyces hyaluronidase, or untreated control chondrocytes were incubated with biotinylated DNA oligonucleotides of 54 select transcription factor binding element sequences (TransSignal Array; Panomics) (Lam and Li, 2002; Zeng et al., 2003). After isolation of protein/DNA complexes, the samples were denatured and retained binding elements hybridized to membranes containing complementary sequences. Hybridized biotinylated oligonucleotides were visualized using ECL reagents. Each transcription factor was quantified in duplicate at 1x concentration, and in duplicate at 0.1x concentration, per condition.
Stimulation of Smad-binding element promoter activity in chondrocyte by BMP-7
An SBE-driven luciferase reporter plasmid PGL3ti-(SBE)4 (Jonk et al., 1998; Althini et al., 2003) was obtained from Dr. Bart Eggen (University of Groningen, Groningen, Netherlands). The immortalized human chondrocyte C-28/I2 cells (Tan et al., 2003) were provided by Dr. Mary Goldring (Harvard Institutes of Medicine, Boston, MA). For transfection, the cells were plated 24 h before transfection at a density of 105 cells/well in 12-well plates, cultured for 24 h, and transiently cotransfected in serum-free media with 1 µg of the PGL3ti-(SBE)4 with FuGENE 6 reagent (Roche). After 24 h the media was changed to DMEM and 0.5% FBS. 48 h after transfection, chondrocytes were treated for 24 h with 100 ng/ml BMP-7 in the absence or presence of Streptomyces hyaluronidase (5 U/ml, 2-h pretreatment followed by a second addition of enzyme at the time of addition of BMP-7), Streptomyces hyaluronidase only for 24 h or no treatment for 24 h. After the treatments the cells were lysed with passive lysis buffer and luciferase activity measured using the dual luciferase assay system (Promega). pSV-ß-galactosidase control vector (Promega) activity was measured in all experiments to normalize for transfection efficiency. Graph presented shows mean value ± SEM based on the analysis of triplicate wells from three experiments.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants P50-AR39239, RO1-AR43384 (W. Knudson), RO1-AR39507 (C.B. Knudson) and grants from the Arthritis Foundation.
Submitted: 25 February 2004
Accepted: 10 August 2004
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References |
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