Fig 1: Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser206 and Ser216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser37, Ser45, or Ser47, because a SDC1 core protein is modified with CS chains according to our previous study (Nadanaka et al., 2018). Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (-) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (-) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells (n = 4, each) was examined by CytoTox-ONETM Assay. (H) The effect of knockdown of ß-catenin on the proliferation of BT-549 cells (n = 6) was investigated. Statistical significance was determined using Student’s t-test.
Fig 2: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (-) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (-) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector (n = 4) and SDC1(NTF)-3xFLAG (n = 3), or proliferation of BT-549 cells overexpressing the empty vector (n = 5), SDC1(FL)-3xFLAG (n = 5), and SDC1(CTF)-3xFLAG (n = 5) was measured.
Fig 3: Enhancement of cell proliferation through the SUMOylation of AKT1 upregulated by the expression of the C-terminal fragment of SDC1. (A) The effect of GM6001 on the proliferation of BT-549 cells overexpressing the empty vector (n = 5), SDC1(FL)-3xFLAG (n = 5), and SDC1(CTF)-3xFLAG (n = 5) was examined. (B) The effect of tannic acid on the proliferation of BT-549 cells overexpressing the empty vector (n = 4), SDC1(FL)-3xFLAG (n = 4), and SDC1(CTF)-3xFLAG (n = 4) was examined. (C) SUMOylated proteins in BT-549 cells overexpressing the empty vector and SDC1(CTF)-3xFLAG were captured using the SUMO-QAPTURE-T kit, and subject to immunoblotting using the anti-Akt1 and anti-SUMO1 antibodies.
Fig 4: Expression of BDNF receptors in human placental tissues. (A) Representative confocal immunofluorescence staining images of showing DAPI (blue) and SDC1 (a syncytiotrophoblast biomarker, green) along with TrkB (red) and p75NTR (red) receptors in the first trimester placenta. (B) Confocal immunofluorescence staining images showing DAPI, SDC1, TrkB, and p75NTR in the term placenta.
Fig 5: C4ST-1 controls BT-549 cell proliferation by regulating the cleavage of SDC1 by MMPs. SDC1 expressed at the cell surface is cleaved by MMPs, and the N-terminal and the C-terminal fragments of SDC1 are generated. Both fragments, SDC1(NTF) and SDC1(CTF), have a positive effect on cell proliferation, and cell proliferation is more affected by SDC1(CTF) than by SDC1(NTF). SDC1(CTF) is involved in SUMOylation of AKT1, although the mechanism underlying SUMOylation by SDC1(CTF) remains unclear. SUMOylation of AKT1 enhances proliferation through S6 kinase signaling pathway. In C4ST-1KO cells, the cleavage of SDC1 by MMPs is suppressed because of reduced expression of MMPs. SUMOylation of AKT1 is inhibited because of decreased SDC1(CTF), and proliferation is slowed down.
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