Fig 1: ADAMTS6 but not ADAMTS10 is processed and catalytically active, binds microfibrillar proteins.(a) Western blot against V5 tag of ADAMTS10 (ATS10 WT) and ADAMTS6 (ATS6 WT) overexpressed proteins using lentivirus. The SDS-PAGE was run under reducing conditions. Both full-length molecules expressed well in this system. Unprocessed ADAMTS10 was present in medium and cell lysates, whereas ADAMTS6 was processed in medium (black arrows). Some larger bands may be aggregated forms of the processed molecule (red arrow), which are possibly trimeric and tetrameric in nature. (b) Full-length LTBP-1 was treated with full-length recombinant ADAMTS6 overnight at 37 °C (molar ratio LTBP-1:ADAMTS6 3:1). Western blot analysis using anti-LTBP-1 C-terminal antibody showed a reduction in full-length LTBP-1 with a relative molecular mass of 185 kDa, and an increase in intensity of a 111 kDa degradation product. The cleavage site of BMP-144, indicated by ‘X’ on LTBP-1 domain map (Fig. 6F), also generates a 110 kDa C-terminal fragment. Control lane (con) contains ADAMTS6 only. (c–f) Biacore sensorgrams showing that: (c) Full-length ADAMTS10 binds the fibrillin-1 PF1 fragment, and (d) full-length ADAMTS6 binds to recombinant N-terminal fibrillin-1 (PF3 and PF4). (e) To further resolve the binding regions, it was found that ADAMTS6 CT regions (Fig. 4) bound fibrillin-1 Ex6–11 fragment16. The binding of C-terminal ADAMTS6 was mapped to exons 6–8 of FBN1, indicated in red. (f) C-terminal ADAMTS6 binds to LTBP-1 C-terminal region (indicated by box). Binding was analysed using Surface Plasmon Resonance, and the response difference (Resp. Diff.) for each experiment was plotted against time (s). Resp. Diff. is the ligand-immobilized flow cell minus the control flow cell.
Fig 2: FBN1 fibers produced in vitro by MT1‐KO MDFs. (a) Confluent cultures of control MDFs transfected with empty vector (a1) and MT1‐KO MDFs transfected with either empty vector (a2), MT1‐MMP‐expression vector (a3), or RECK‐expression vector (a3) were subjected to double immunofluorescence staining with anti‐FBN1 (red) and anti‐FN (green) antibodies followed by nuclear counterstaining (blue). Scale bar = 10 μm. Note the poor formation of both FBN1 and FN fibers in MT1‐KO MDFs (a2), the ability of MT1‐MMP‐expression vector to at least partially restore the formation of FBN1 and FN fibers (a3), and the ability of RECK to restore the formation of FBN1 fibers to some extent but not the formation of FN fibers (a4). Similar results were obtained in three independent experiments. (b and c) Properties of FBN1 and FN fibers. (b) The intensity of FBN1 (b1) and FN (b2) signals at peaks (left panels) and valleys (right panels). The number of data are as follows. FBN1‐Peaks: Cont. + Vector, 1,375; MT1‐KO + Vector, 1,426; MT1‐KO + MT1, 1,444; MT1‐KO + RECK, 1,302. FBN1‐Valleys: Cont. + Vector, 1,367; MT1‐KO + Vector, 1,416; MT1‐KO + MT1, 1,435; MT1‐KO + RECK, 1,292. FN‐Peaks: Cont. + Vector, 1,406; MT1‐KO + Vector, 1,460; MT1‐KO + MT1, 1,295; MT1‐KO + RECK, 1,472; FN‐Valleys: Cont. + Vector, 1,395; MT1‐KO + Vector, 1,453; MT1‐KO + MT1, 1,282; MT1‐KO + RECK, 1,462. (c) Width of FBN1 fibers (c1) and FN fibers (c2). Welch's t test: *p < .05, **p < 5 × 10−3, ***p < 5 × 10−10. Note that the mean of widths (indicated by X in c) is largely consistent with the above observations of immunofluorescence images (a). Similar results were obtained in three independent experiments. Cont. control; FN, fibronectin; KO, knockout; M, MT1‐MMP‐expression vector; MDF, mouse dermal fibroblast; MT1‐MMP, membrane‐type 1‐ matrix metalloproteinase; R, RECK‐expression vector; V, empty vector
Fig 3: FBN1 and FN fibers produced in vitro by MDFs derived from wild-type (WT) and RECK-Hypo mice. (a) Confluent cultures of MDFs prepared from WT (a1) or Reck-Hypo (a2) mice at Postnatal Day 3 were subjected to double immunofluorescence staining with anti-FBN1 (red) and anti-FN (green) antibodies followed by nuclear counterstaining (blue). Images of the same field in each color and the merged image are shown in four-parts panels. Images of background staining (i.e., no primary antibodies) are also shown (a3). Scale bar = 10 µm. As compared to the control, FBN1 fibers produced by Reck-Hypo MDFs (a2) tend to form local aggregates in the area where FN fibers are few (yellow arrows). Substantial FN signals are found in spotty structures in RECK-Hypo MDFs (green signals in a2). (b and c) Properties of FBN1 and FN fibers. (b) Intensity of FBN1 (b1) and FN (b2) signals at peaks (P) and valleys (V). (b1) Number of data: WT P, 1,134 and WT V, 1,123; Hypo P, 1,149 and Hypo V, 1,139. Student's t test (WT vs. Hypo): P, p = .0015; V, p = .0013. (b2) Number of data: WT P, 1,148; WT V, 1,143; Hypo P, 1,122; Hypo V, 1,110. Student's t test (WT vs. Hypo): P, p = .094; V, p = .041. (c) Width of FBN1 fibers (c1) and FN fibers (c2). Welch's t test (WT vs. Hypo): FBN1, p = .043; FN, p = .13. Similar results were obtained from three different mice of each group. FN, fibronectin; MDF, mouse dermal fibroblast
Fig 4: FBN1 and FN fibers produced by the human osteosarcoma cell line MG63 with or without RECK expression. (a) Confluent cultures of a RECK-deficient MG63 subline (m1) stably transfected with empty vector (a1) or RECK-expression vector (a2) were subjected to double immunofluorescence staining. Images of background staining (i.e., no primary antibodies) are also shown (a3). Scale bar = 10 µm. Note that FBN1 fibers (red) and FN fibers (green) are thicker and forming a more elaborate network when RECK is expressed (a2 vs. a1). (b and c) Properties of FBN1 and FN fibers. (b) The intensity of FBN1 (b1) and FN (b2) signals at peaks (P) and valleys (V). (b1) Number of data: Vector P, 1,651 and Vector V, 1,641; RECK P, 1,722 and RECK V, 1,709. Welch's t test (Vector vs. RECK): P, p = 8.87 × 10-44; V, p = 3.36 × 10-44. (b2) Number of data: Vector P, 1,803 and Vector V, 1,791; RECK P, 1,708 and RECK V, 1,697. Welch's t test (WT vs. Hypo): P, p = 9.68 × 10-73; V, p = 3.35 × 10-62. (c) Width of FBN1 fibers (c1) and FN fibers (c2). Welch's t test (Vector vs. RECK): FBN1, p = 8.58 × 10-14; FN, p = 6.59 × 10-7. Note the increased peak intensity and its variation (pale blue box vs. blue box in b) as well as fiber width and its variation (orange box vs. blue box in c) for FBN1 and FN fibers in RECK-expressing cells. Ab, antibody; FN, fibronectin; KO, knockout; WT, wild-type
Fig 5: Immunoblot detection of RECK, integrins, FN, and FBN in RECK-KO MG63 mutants. Lysates of MG63 cells (Lanes 1 and 2) and two RECK-deficient sublines, m1 (Lanes 3 and 4) and m2 (Lanes 5 and 6), transfected with either empty vector (V; Lanes 1, 3, and 5) or RECK-expression vector (R; 2, 4, and 6) were subjected to immunoblot assay with antibodies against RECK (a), integrin ß1 (b), integrin a2 (c), integrin a5 (d), FN (e), and FBN (f). Symbols: #, full-length protein; red arrowhead, a fragment more abundant when RECK is absent; and green arrow, a fragment more abundant when RECK is present. (f) The full-length FBN bands (312–315 kDa) are undetectable under these conditions. (g–k) Effects of RECK on fragments of integrins, FN, and FBN. (b–f) Immunoblot images together with similar data obtained using two other RECK-deficient lines (data not shown) were subjected to densitometry. The ratio of intensity between each integrin subfragment and the full-length integrin band (g–i) and the relative intensity of FN (j) and FBN (k) normalized against GAPDH were determined. The graph represents the mean ± standard error of the mean. Student's t test: *p < .05. (l) Immunoblot detection of FBN fragments in MG63 conditioned media. Confluent cultures of the indicated transfectants were exposed for 3 hr to the same volume of conditioned medium prepared from the control cells (Lanes 1, 3, 5, 7, 9, and 11) or ADAMTS10-expressing cells (Lanes 2, 4, 6, 8, and 10), and then the culture supernatants were subjected to immunoblot assay using anti-FBN antibodies. (m) The data were subjected to densitometry, and the total density of three bands (arrowheads) are presented. Note that the amount of FBN fragments released from the cells is higher in the absence of RECK (Lanes 5, 6 vs. 7, 8; Lanes 9, 10 vs. 11, 12) and that in the absence of RECK, the addition of ADAMTS10 to the cells increases the amount of FBN fragments released from the cells (Lanes 6, 10 vs. 5, 9). FBN, fibrillin; FN, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout
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