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* Department of Surgery, McGill University, Montreal, Quebec, Canada;
McGill University and Genome Quebec Innovation Center, Montreal, Quebec, Canada; and
Département de Stomatologie, Université de Montréal, Quebec, Canada
4Correspondence: McGill University, Montreal General Hospital, 1650 Cedar Ave., Rm. C9–177, Montreal, Quebec H3G 1A4, Canada. E-mail: anie.philip{at}mcgill.ca
SPECIFIC AIMS
The multifunctional nature of TGF-ßbeta; implicates a requirement for strict regulation of its cellular signaling. This regulation is achieved at multiple levels of the signaling pathway, including modulation of signaling receptor activity at the cell surface by other TGF-ßbeta; binding proteins such as betaglycan and endoglin. Several groups, including ours, have reported the occurrence of cell surface glycosylphosphatidylinositol (GPI)-anchored proteins that bind TGF-ßbeta; in an isoform-specific manner. We have recently shown that human keratinocytes defective in GPI-anchor synthesis show enhanced TGF-ßbeta; responses, suggesting that GPI-anchored proteins regulate TGF-ßbeta; signaling. Our results implicated a specific cell surface 150 kDa GPI-anchored TGF-ßbeta;1-binding protein, which we designated as r150, in modulating TGF-ßbeta; signaling in keratinocytes. The aims of the present study were to determine the molecular identity of r150 and to assess its potential as a regulator of TGF-ßbeta; signaling and responses in keratinocytes.
PRINCIPAL FINDINGS
1. Molecular identification of r150 as CD109
To determine the molecular identity of r150, the human keratinocyte cell line HaCaT was treated with phosphatidylinositol specific-phospholipase C (PIPLC) to release GPI-anchored proteins from the cell surface, and the supernatant containing these proteins was purified on a TGF-ßbeta;1 affinity column. The affinity-purified fractions containing the 150 kDa protein were resolved by SDS-PAGE, and the 150 kDa bands were excised and analyzed by mass spectrometry. This yielded a microsequence of 19 amino acids, SNLIQQWLSQQSDLGVISK, which matched to the human CD109 sequence. The encoded protein has 1445 amino acids with an N-terminal signal peptide of 21 amino acids and a C-terminal consensus GPI-anchor signal sequence with the cleavage predicted to occur at amino acid 1420. CD109 belongs to the 
2-macroglobulin (2M)/complement superfamily and shares several structural motifs with members of that gene family, including a putative bait region (aa 651–683), a thioester signature motif (aa 918–924), and a thioester reactivity defining hexapeptide (aa 1030–1035).
2. CD109 binds TGF-ßbeta;1 and forms a heteromeric complex with the TGF-ßbeta; signaling receptors
The ability of CD109 to bind TGF-ßbeta;1 was demonstrated using affinity labeling of HaCaT cells overexpressing CD109 or HaCaT cells in which CD109 expression was blocked with antisense morpholino oligos (data not shown). We then determined whether CD109 forms a heteromeric complex with the TGF-ßbeta; signaling receptors in the presence of TGF-ßbeta; using affinity labeling/coimmunoprecipitation analyses. Figure 1
shows that membrane extracts of HaCaT cells not immunoprecipitated (NIP) displayed the characteristic TGF-ßbeta; receptor profile with type I (RI) and II (RII) receptors, and r150/CD109 (lane 1). The anti-CD109 antibody (Ab), 1B3, immunoprecipitated endogenous r150, and coimmunoprecipitated detectable amounts of RI and RII, indicating association of r150/CD109 with the TGF-ßbeta; signaling receptors (lane 2). The reciprocal experiment demonstrating the coimmunoprecipitation of r150/CD109 with an anti-RI Ab (lane 3) confirms that CD109 associates with RI in the presence of ligand.
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We next examined whether r150/CD109 associates with the TGF-ßbeta; signaling receptors in the absence of TGF-ßbeta; using immunoprecipitation in tandem with Western blot analysis. HaCaT cells were washed with mild acid to remove endogenous TGF-ßbeta; and membrane extracts immunoprecipitated with specific anti-TGF-ßbeta; receptor antibodies, were analyzed by Western blot with an anti-CD109 Ab. Coimmunoprecipitation of CD109 was detectable in appreciable amounts with the anti-RI Ab, and in somewhat lower amounts with anti-RII and anti-RIII antibodies (data not shown). These results demonstrate that CD109 associates with the TGF-ßbeta; signaling receptors and betaglycan in the absence of ligand, and at physiological receptor concentrations.
The above results indicate that the anti-RI Ab coimmunoprecipitates substantial amounts of CD109 protein, raising the possibility that RI and CD109 might interact directly. To explore this possibility, we in vitro translated CD109 and examined its ability to bind recombinant glutathione (GSH) S-transferase (GST)-RI, as compared to GST, in vitro. We found that in vitro translated [35S]CD109 is specifically retained by GST-RI, but not GST, immobilized to GSH-sepharose beads. These data suggest that CD109 interacts directly with the type I TGF-ßbeta; receptor.
3. CD109 inhibits TGF-ßbeta;1 signaling and responses in human keratinocytes
TGF-ßbeta;1-induced phosphorylation of Smad2 and Smad3
Activation of Smad2 and Smad3 by receptor mediated phosphorylation is a central event in TGF-ßbeta; signal transduction. We, therefore, determined the effect of CD109 on TGF-ßbeta;1-induced phosphorylation of Smad2 and Smad3. Overexpression of CD109 in HaCaT cells led to a marked decrease in TGF-ßbeta;1-induced phosphorylation of Smad2 and Smad3, while total levels of Smad2 and Smad3 remained unaltered (data not shown). Furthermore, blocking CD109 expression in HaCaT cells resulted in enhanced TGF-ßbeta;1-induced phosphorylation of Smad2 and Smad3 (data not shown). Together, these results indicate that CD109 negatively modulates TGF-ßbeta;1-induced phosphorylation of Smad2 and Smad3.
TGF-ßbeta;1-induced transcriptional activity
We then explored the effect of CD109 on TGF-ßbeta;1-induced transcriptional activity, using two TGF-ßbeta;-responsive luciferase reporter constructs, 3TP-lux and (CAGA)12-lux. Figure 2
A shows that overexpression of CD109 in HaCaT cells led to a significant (P<0.05) reduction in basal and TGF-ßbeta;1-induced 3TP-lux activity compared with empty vector (EV) transfectants. Similar results were obtained in 293 cells and mouse embryonic fibroblasts (data not shown). Furthermore, overexpression of CD109 in HaCaT cells led to a marked reduction in TGF-ßbeta;1-induced (CAGA)12-lux activity (data not shown)
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TGF-ßbeta;1-induced extracellular matrix (ECM) deposition
Since TGF-ßbeta; is a key regulator of ECM synthesis and breakdown, we examined whether CD109 regulates the levels of plasminogen activator inhibitor-1 (PAI-1) or fibronectin. Figure 2B
shows that overexpression of CD109 led to 2.5- and 7.5-fold decreases in PAI-1 levels at 5 pM and 50 pM TGF-ßbeta;, respectively (upper panel), and 2- and 3-fold decreases in fibronectin levels at 5 pM and 50 pM TGF-ßbeta;, respectively (middle panel), when compared to empty vector (EV) transfectants. Western blot for actin demonstrated that equivalent amounts of protein were loaded in each lane (lower panel).
TGF-ßbeta;1-induced growth inhibition and in vitro wound closure
TGF-ßbeta; shows both a potent antiproliferative effect and pro-migratory effect on human keratinocytes. We, therefore, sought to determine whether CD109 could modulate these effects. HaCaT cells stably transfected with CD109 showed a significant reduction in TGF-ßbeta;1-induced thymidine incorporation and in vitro wound closure (data not shown). Furthermore, blocking expression of CD109 using antisense morpholino oligonucleotides had the opposite effect on TGF-ßbeta;1-mediated growth inhibition (data not shown). These results suggest that CD109 inhibits TGF-ßbeta;1 whole cell responses in human keratinocytes.
4. CD109 inhibits TGF-ßbeta; signaling independently of ligand sequestration
Our data demonstrate that CD109 associates with the TGF-ßbeta; signaling receptors in the presence or absence of TGF-ßbeta;1 ligand, raising the possibility that CD109 may exert its effect on TGF-ßbeta;1 signaling by directly modulating receptor activity. However, since our previous work and the present study demonstrate that CD109 binds TGF-ßbeta;1, it was of interest to test whether CD109 exerts its effect by sequestering TGF-ßbeta;1 ligand away from the signaling receptors. We found that increasing the concentration of CD109 on the cell surface increases TGF-ßbeta;1 binding to CD109, but does not alter TGF-ßbeta;1 binding to the type I and II receptors (data not shown), suggesting that CD109 does not sequester ligand away from the receptors. Furthermore, we also found in 293 cells that CD109 inhibits transcriptional activity induced by a constitutively active type I receptor (T204D) in the absence of ligand (P<0.05) (data not shown). Virtually identical results were obtained in the presence of a TGF-ßbeta; neutralizing Ab (1D11, 10 µg/ml) (data not shown), suggesting that the contribution of autocrine TGF-ßbeta; to transcriptional activation is negligible in these cells. These data suggest that CD109 can inhibit TGF-ßbeta;1 signaling independently of ligand sequestration.
CONCLUSIONS AND SIGNIFICANCE
Several groups including ours have reported the occurrence of cell surface GPI-anchored proteins that bind TGF-ßbeta; in an isoform-specific manner. However, the identities of these proteins were not known. In the present study, we demonstrate that r150, a GPI-anchored TGF-ßbeta;1-binding protein that we previously described on keratinocytes, represents CD109, a novel member of the 2M/complement superfamily. We demonstrate that CD109 is a component of the TGF-ßbeta; receptor system and a negative regulator of TGF- ßbeta;1 signaling and responses in human keratinocytes (Fig. 3
). In addition, our data suggest that CD109 can inhibit TGF-ßbeta; signaling independently of ligand sequestration and may exert its effect on TGF-ßbeta; signaling by direct modulation of receptor activity. Together, these results suggest that CD109 plays a unique role in the regulation of isoform-specific TGF-ßbeta; signaling in keratinocytes. The present study represents the first report on the molecular identification of a GPI-anchored TGF-ßbeta; binding protein.
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FOOTNOTES
1 These authors contributed equally to this work. 
2 Present address: TargeGen Inc., 9380 Judicial Dr., San Diego 92121, CA, USA.
3 Present address: Dept. of Colorectal Surgery, Tianjin Medical University Cancer Hospital, Tianjin, P.R. China.
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5229fje
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