Determination of the CD148-Interacting Region in Thrombospondin-1

<div><p>CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1^st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1^st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1^st, but not the 2^nd and 3^rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1^st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1^st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1^st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis <i>in vivo</i>. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1^st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.</p></div

A CD148-interacting trimeric TSP1 fragment inhibits endothelial cell proliferation and angiogenesis.

<p><b>(A)</b> HRMEC cells were treated with the trimeric TSP1 fragment (1.5, 3.0, 6.0, 12.0 nM) containing the procollagen domain and the 1^st type 1 repeat or whole TSP1 (12 nM) and its effects on cell proliferation were assessed (left panel). The effects of CD148 knockdown were also assessed with 12 nM of the TSP1 fragment (right panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: CD148 knockdown largely attenuates the activity of the TSP1 fragment to inhibit cell proliferation of HRMEC cells. <b>(B)</b> Upper panels: Gelfoam sponges loaded with vehicle or 100 ng VEGF plus or minus 100 pmol of the trimeric TSP1 fragment containing the procollagen domain and the 1^st type 1 repeat were subcutaneously implanted into the dorsal flank of either wild-type or CD148 knockout mice. At day 7, the mice were injected intravenously with 2% TRITC-dextran to label vessels, then the sponges were excised for analysis. Left panels show representative results of whole sponges under a fluorescence microscope. Right panel shows the TRITC-based quantification of vessel density in whole sponges. TRITC-positive pixel area was measured. Data show mean ± SEM of six sponges from independent mice. ** <i>P</i> < 0.05, *** <i>P</i> < 0.01 Note: A CD148-interacting trimeric TSP1 fragment inhibits VEGF-induced angiogenesis in wild-type, but not CD148 knockout, mice. Lower panels: Paraffin sections were processed from each sponge. Vessel density was assessed by vWF immunostaining. The sections were counterstained with DAPI. Left panels show representative results of vWF immunostaining in each condition. Right panel shows FITC-based quantification of vessel density in each group. Data show mean ± SEM of six sponges from independent mice. ** <i>P</i> < 0.05, *** <i>P</i> < 0.01</p

The 1^st type 1 repeat is required for TSP1/CD148-mediated cell growth inhibition.

<p><b>(A)</b> The trimeric TSP1 fragment (ΔType1-R1) that contains the procollagen domain and the 2^nd and 3^rd, but not the 1^st, type 1 repeat was prepared using HEK293E cells. Upper panel shows a schematic representation of the trimeric TSP1 fragment that lacks the 1^st type 1 repeat. Amino acid residues (aa 374–429) of the 1^st type 1 repeat were deleted. Lower panel shows colloidal blue staining of the purified ΔType1-R1 fragment. Five micrograms of protein were separated on a 10% polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions and stained with colloidal blue to assess size, purity, and trimerization. The expected size of protein is also shown. <b>(B)</b> A431D/CD148wt or A431D/CD36 (stably expressing CD36) cells were treated with 12 nM of trimeric TSP1 fragments that lack or contain the 1^st type 1 repeat or whole TSP1 protein. Cell density was measured at the indicated time points. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. Note: The ΔType1-R1 fragment shows no growth inhibitory activity in A431D/CD148wt cells, while it inhibits cell proliferation in A431D/CD36 cells. <b>(C)</b> A431D/CD36 cells were treated with trimeric TSP1 fragments (12 nM) that lacked or contained the 1^st type 1 repeat or whole TSP1 protein (12 nM) for 18 h. Tyrosine phosphorylation of p38 and cleaved caspase 3 was assessed by immunoblotting using the phopho-specific p38 (pThr180+Tyr182) or cleaved caspase 3 antibodies. The membranes were reprobed with antibodies to total p38 or γ-tubulin. Representative data of four independent experiments is shown. <b>(D)</b> A series of monomeric TSP1 fragments were prepared from the regions containing the procollagen domain and type 1 repeats as shown in a schema on right side. Each fragment (17 nM) was incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone), and Fc-proteins were pulled down with protein-G beads. Bound TSP1 fragments were assessed by anti-Myc immunoblotting (upper panel). Half of each sample was subjected to anti-CD148 immunoblotting to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: TSP1 fragments that contain the 1^st type 1 repeat bind to CD148-Fc. <b>(E)</b> A431D/CD148wt cells were treated with or without indicated TSP1 fragments (36 nM) for 1 h, then a trimeric TSP1 fragment (12nM) containing the procollagen domain and type 1 repeats was added to the medium. Cell proliferation was assessed at day 2. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: Only the 1^st type 1 repeat blocks cell growth inhibition induced by the trimeric TSP1 fragment.</p

Type 1 repeats are required for TSP1/CD148-mediated cell growth inhibition.

<p><b>(A)</b> Trimeric TSP1 fragments containing the procollagen domain and either all three, two, one, or none of type 1 repeats were prepared using HEK293E cells. Left panel shows a schematic representation of the trimeric TSP1 fragments used in this study. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Two micrograms of protein were separated on a 4–20% gradient polyacrylamide gel in reducing (+DTT) and non-reducing (-DTT) conditions, then stained with colloidal blue to assess its size, purity, and trimerization. The expected size of protein is also shown. <b>(B)</b> A431D cells stably expressing wild-type CD148 (A431D/CD148wt cells) were plated in 96-well plates, starved, and treated with 12 nM of trimeric TSP1 fragments or whole TSP1 protein. Cell density was measured at the indicated time points (left panel). The dose dependency of the effects was also evaluated at day 2 (right panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. <b>(C)</b> The effects of a trimeric TSP1 fragment (6.0 nM) containing the procollagen domain and the 1^st type 1 repeat on cell proliferation of A431D (lacking CD148) and A431D/CD148cs (stably expressing a catalytically inactive form of CD148) cells are shown. Cell proliferation was assessed as in (B). The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** <i>P</i> < 0.05 <b>(D)</b> CD148 was knocked down in A431D/CD148wt cells using the lentivirus encoding CD148-targeting shRNA (shRNA #1, shRNA #2). The lentivirus encoding scrambled shRNA was used as a control. The cells were subjected to a cell proliferation assay and the effects of CD148 knockdown on growth inhibition of a trimeric TSP1 fragment (12 nM) containing the procollagen domain and the 1^st type 1 repeat were assessed. The data show mean ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** <i>P</i> < 0.05 Note: CD148 knockdown largely attenuates the TSP1 fragment’s growth inhibitory activity in A431D/CD148wt cells.</p

Assessment of CD148-interacting region in TSP1.

<p><b>(A)</b> Recombinant TSP1 fragments that correspond to the structural elements were prepared using HEK293E cells. Left panel shows a schematic representation of the TSP1 fragments. The number of amino acid residues includes the signal peptide sequence. Right panel shows colloidal blue stain of the purified TSP1 fragments. Twelve micrograms of protein were separated on a 10% polyacrylamide gel and stained with colloidal blue to assess size and purity. The expected size of protein is also shown. <b>(B)</b> TSP1 fragments (17 nM) were incubated with either 44 pmol of CD148-Fc or control Fc (Fc alone). Fc-proteins were pulled down with Protein-G beads and the binding of TSP1 fragments was assessed by immunoblotting using anti-Myc antibody (upper panel). The membrane was reprobed with anti-CD148 antibody to confirm the pull down of CD148-Fc (lower panel). Representative data of five independent experiments is shown. Note: The TSP1 fragment containing the procollagen domain and type 1 repeats binds to CD148-Fc. <b>(C)</b> Protein-A plates conjugated with CD148-Fc (11.3 nM) or equal molar of control Fc were incubated with AP-TSP1 or AP (12 nM) in the presence or absence of TSP1 fragments (25 nM) or whole TSP1 protein (25 nM). The bound AP-TSP1 was assessed by an AP activity assay. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 Note: The binding of AP-TSP1 to CD148-Fc is blocked with either a TSP1 fragment containing the procollagen domain and type 1 repeats or whole TSP1 protein.</p

Trimeric TSP1 fragments that contain the 1^st type 1 repeat increase CD148 catalytic activity and reduce tyrosine phosphorylation of EGFR and ERK1/2 in A431D/CD148wt cells.

<p><b>(A)</b> Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. CD148 was immunoprecipitated using anti-CD148 antibody or class-matched control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO₄). The amount of CD148 in the immunocomplexes was evaluated by immunoblotting using anti-CD148 antibody (lower panel). The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 vs. vehicle-treated cells. Right: To assess the specificity of the effect, a trimeric TSP1 fragment containing the procollagen domain and the 1^st type 1 repeat was added to A431D/CD148wt cells with 11.3 nM of CD148-Fc or control Fc (Fc alone), then CD148 catalytic activity was assessed as in left panel. The data show mean ± SEM of quadruplicate determinations. Representative data of five independent experiments is shown. ** <i>P</i> < 0.05 vs. vehicle-treated cells. Note: CD148-Fc, but not control Fc, abolishes the activity of the TSP1 fragment to increase CD148 catalytic activity. <b>(B)</b> Left: A431D/CD148wt cells were treated with the indicated trimeric TSP1 fragments (12 nM) or whole TSP1 protein (12 nM) for 15 min. Tyrosine phosphorylation of EGFR (immunoprecipitated) and ERK1/2 was assessed by immunoblotting using the phopho-specific EGFR (Y1173) or ERK1/2 (T202/Y204) antibodies. The membranes were reprobed with antibodies to total EGFR or ERK1/2. Representative data of four independent experiments is shown. Right: A431D and A431D/CD148cs cells were treated with a trimeric TSP1 fragment (12 nM) that contains the procollagen domain and the 1^st type 1 repeat or whole TSP1 protein (12 nM), and tyrosine phosphorylation of EGFR (immunoprecipitated) and ERK1/2 was assessed as in left panel. Representative data of four independent experiments is shown. Note: No effects are observed in A431D and A431D/CD148cs cells.</p

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

<div><p>CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin <i>in vitro</i>. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.</p></div

CD148 regulates the tyrosine phosphorylation of p120, β-catenin, and Src upon E-cadherin engagement.

<p>Effects of CD148 in the cadherin adhesion-associated tyrosine phosphorylation of p120, β-catenin, and Src were assessed by a calcium-switch assay and immunoblot analysis using A431D/E-caherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells. For p120 and β-catenin, tyrosine phosphorylation of p120 and β-catenin that were co-immunoprecipitated with E-cadherin was assessed by immunoblotting. In A431D/E-cadherin 764 AAA cells, p120 was immunoprecipitated. The membranes were reprobed with p120, β-catenin and Src antibodies and a ratio of phosphorylated to total protein was quantified by densitometry. Data are representative of five independent experiments. CD148 WT, but not CS, reduces the tyrosine phosphorylation of p120, β-catenin, and Src (Y529) upon E-cadherin engagement in A431D/E-cadherin WT cells, while it increases the phosphorylation of Y228 (a Src site) in p120. These effects are not observed in A431D/E-cadherin 764 AAA cells.</p

CD148 knockdown reduces cell-cell adhesion and E-cadherin contacts accompanied by a decrease in Rac1 activity in A431 cells.

<p><b>A)</b> A431 cells was infected with a lentivirus encoding CD148-targeting or scrambled shRNA. Cells were harvested at 72 h after infection and the expression of CD148, E-cadherin, p120, and β- catenin was assessed by immunoblot analysis. Equal loading was confirmed by reblotting the membrane for β-actin. The targeting shRNA reduces CD148 expression (∼65%) without altering the E-cadherin, p120, and β-catenin expression. <b>B)</b> CD148 knock-down cells and the control cells treated with scrambled shRNA were subjected to a handing drop assay. Data are representative of five independent experiments. CD148 knockdown reduces cell aggregation in A431 cells. <b>C and D)</b> CD148 knock-down and control cells were subjected to a calcium switch assay and were immunostained for E-cadherin (panel C). Rac1 activity in these cells was also assessed (panel D). Representative results of four independent experiments are shown. The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148-targeting cells. CD148 knockdown reduces the E-cadherin contact formation accompanied by a decrease in Rac1 activity.</p

Introduction of CD148 forms to A431D and A431D/E-cadherin cells and its effects for E-cadherin and catenin expression and complex formation.

<p><b>A)</b> Wild-type (WT) or catalytically inactive (CS) CD148 was stably introduced to A431D cells lacking classical cadherins [E-cad (−)] or expressing wild-type (WT) or p120-uncoupled mutant (764AAA) E-cadherin. The expression levels of CD148 in these cells were examined by immunoblotting (upper panel) and flow cytometry (lower panel). The loading was assessed by reblotting the membrane for β-actin. <b>B)</b> The levels of E-cadherin, p120, and β-catenin in nearly confluent CD148 stable cells were assessed by immunoblotting, comparing with CD148-negative cells (upper panels). The formation of E-cadherin/catenin complex was assessed by co-immunoprecipitation with E-cadherin (lower panels). Note: The association of E-cadherin with p120 is not observed in A431D/E-cadherin 764 AAA cells.</p