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First published online 12 August 2003
doi: 10.1242/jcs.00699

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ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating ß1 integrin function

Nobuko Kawaguchi^1,*, Christina Sundberg^1,*, Marie Kveiborg¹, Behzad Moghadaszadeh¹, Meena Asmar¹, Nikolaj Dietrich¹, Charles K. Thodeti¹, Finn C. Nielsen², Peter Möller³, Arthur M. Mercurio⁴, Reidar Albrechtsen¹ and Ulla M. Wewer^1,

¹ Institute of Molecular Pathology, University of Copenhagen, Frederik's vej 11, 2100 Copenhagen, Denmark
² Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark
³ Department of Pathology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany
⁴ Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

View larger version (85K): [in a new window]   Fig. 1. ADAM12 expression in adipose tissues. (A-D) ADAM12 expression was demonstrated by immunostaining (arrows) in white adipose tissue from a normal mouse (A), in a human lipoma (B), in mouse brown and white adipose tissue (C), and in a human hibernoma (D). (E) The expression of ADAM12 mRNA in mouse abdominal (Ab)-, epididymal (Ep)-white adipose tissue (WAT) and brown adipose tissue (BAT), was shown by northern blot analysis. Normal adult muscle and embryonic muscle (E17) were used as negative and positive controls, respectively. The expression of GAPDH was analyzed as a loading control. The brownish color represents the positive immunostaining reaction product, and the blue color is hematoxylin counterstaining. Bars, 20 µm (A,B) and 30 µm (C,D).

View larger version (50K): [in a new window]   Fig. 2. ADAM12 mRNA expression is transiently upregulated before adipocyte differentiation. (A) Total RNA was extracted from mouse 3T3-L1 cell cultures at various times during differentiation. The levels of ADAM12, PPAR and GAPDH mRNA expression were analyzed by northern blot. GAPDH and rRNA served as loading controls. (B) Quantitative analysis of ADAM12 and PPAR mRNA expression, normalized to GAPDH mRNA levels. (C) Northern blot analysis of ADAM12 RNA extracted from human liposarcoma-derived LiSa-2 cells at various stages of differentiation. (D) ADAM12 protein expression was analyzed by western blot in extracts from growing C3H10T1/2, 3T3-L1 and LiSa-2 cells. CHOK1 cells transfected with control vector and full-length mouse ADAM12 cDNA were used as negative (NC) and positive (PC) controls, respectively. D0, day 0 confluent cells; D3, D4, D9 and D17, days 3, 4, 9 and 17 after the onset of differentiation, respectively; G, growing fibroblastic cells.

View larger version (95K): [in a new window]   Fig. 3. Distinct upregulation of ADAM12 at the cell surface before onset of 3T3-L1 adipocyte differentiation. Growing cells are shown in (A) and (D), confluent (day 0) cells in (B) and (E), and maturing cells after 4 days of differentiation in (C) and (F). (A-C) Adherent cells were immunostained after permeabilization. (D F) Cells were immunostained in suspension without previous permeabilization, then subjected to cytospin centrifugation, as described in Materials and Methods. (G-I) ADAM12 mAbs were added to the cultures for 5 hours (G) 2 days (H) and 6 days (I), followed by detection of the ADAM12:IgG complex with a secondary Ab to visualize endocytosed ADAM12. The central red staining in the cells in panels A and B represent nonspecific nuclear immunoreactivity. Bars, 18 µm (A,B,D,E,G,H) and 30 µm (C,F,I).

View larger version (72K): [in a new window]   Fig. 4. Coordinated localization of fibronectin, actin stress fibers, ß1 integrin and coimmunoprecipitation of ADAM12 and ß1 integrin. Immunostaining of fibronectin (A), F-actin (B) and ß1 integrin (C) in growing LiSa-2 cells. Fibronectin staining outside the cells and actin stress fibers inside the cells are parallel to each other, with the cell-surface ß1 integrin receptor localized at the matrix points. (D) Co-immunoprecipitation of ADAM12 and ß1 integrin from cell extracts of LiSa-2 (lane 3), C3H10T1/2 (lane 5) and 3T3-L1 (lane 7) cells. For this experiment, cell extracts were immunoprecipitated with monoclonal antibodies to ADAM12 (lanes 3,5,7) or control IgG (lanes 2,4,6), and complexes were examined by western blotting with monoclonal antibodies to human ß1 integrin JB1A (lanes 2,3) and to mouse ß1 integrin clone 18 (lanes 4-7). Lane 1 is a direct western blot of ß1 integrin in LiSa-2 cell lysate using the JB1A monoclonal antibody. Bar, 6 µm.

View larger version (65K): [in a new window]   Fig. 5. Reduced ß1 integrin activity upon ADAM12 cell-surface localization. Cell-surface immunostaining of ADAM12 (A,B) and activated ß1 integrin, clone 12G10 (C,D) or total ß1 integrin, clone K20 (E,F) in growing cells (A,C,E) and in confluent day 0 cells (B,D,F). Note the inverse relationship between cell-surface localization of the activated form of ß1 integrin and ADAM12 at the cell surface, while no apparent difference in the total amount of ß1 integrin was detected by immunostaining (compare E and F). This was confirmed by FACS analysis using activated ß1 integrin, clone 12G10 (G) or total ß1 integrin, clone MAR4 (H). Growing cells and day 0 cells were shown with gray and red, respectively. Bars, 25 µm (A,B) and 20 µm (C-F).

View larger version (128K): [in a new window]   Fig. 6. Cell-surface ADAM12 induces actin cytoskeleton reorganization in 3T3-L1 cells. (A-C) 3T3-L1 cells were analyzed by staining with rhodamine-conjugated phalloidin to monitor F-actin organization during differentiation. (D-L) Growing 3T3-L1 cells were transfected with plasmids encoding ADAM12-cyt/EGFP (D-F), ADAM12-cyt catalytic site mutation/EGFP (G-I) or vector control (J-L) and analyzed for EGFP (D,G,J), F-actin (E,H,K) and by phase-contrast microscopy (F,I,L). Cytoskeletal organization was disrupted in cells transfected with either ADAM12-cyt or ADAM12-cyt with a catalytic site mutation (E,H), whereas actin stress fibers were maintained in cells transfected with the vector control (K). Single and double arrows indicate actin stress fibers and cortical actin, respectively. Bars, 10 µm (A-C) and 7 µm (D-L).

View larger version (76K): [in a new window]   Fig. 7. Decreased expression of ADAM12 in 3T3-L1 cells by RNAi inhibits actin cytoskeleton reorganization. 3T3-L1 cells were co-injected with ADAM12 siRNA (A,B) or a scrambled siRNA oligo (C,D) together with a control EGFP vector. Cells were analyzed for EGFP by direct fluorescence microscopy (A,C) or for F-actin by staining with rhodamine-conjugated phalloidin (B,D). Cells injected with ADAM12 siRNA (B) maintained their stress fibers (single arrow), whereas cells injected with scrambled siRNA (D) did not contain stress fibers and showed cortical actin instead (double arrows). As a control siRNA-injected, EGFP-positive cells (E) did not exhibit ADAM12 immunostaining (F), whereas noninjected cells (G) did exhibit ADAM12 immunostaining (H). Bars, 8 µm.

View larger version (68K): [in a new window]   Fig. 8. Focal adhesion formation and cell adhesion are downregulated in 3T3-L1 cells overexpressing ADAM12 at the cell surface. Cells (suspended in A,B and adherent in D-J) with constitutive expression of ADAM12-cyt at the cell surface (A,D,G) and control cells (B,E,H) were examined. (A-C) ADAM12 immunostaining and western blot analysis. (D-F) Staining with rhodamine-conjugated phalloidin to monitor F-actin organization (D,E) showed that ADAM12-cyt-expressing cells were smaller than control cells, and that, compared with the control cells, a significantly higher percentage of cells had fewer than five stress fibers (P<0.02) (F). (G-I) Vinculin immunostaining (G,H) showed that a higher percentage of ADAM12-cyt expressing cells than control cells had fewer than five focal adhesions (P<0.02) (I). (J) Compared with the control cells, cell attachment on fibronectin was reduced (P<0.01) in ADAM12-cyt-expressing cells. Bars, 10 µm (A,B) and 15 µm (D,E,G,H).

View larger version (42K): [in a new window]   Fig. 9. Increased apoptosis and reorganized fibronectin-rich extracellular matrix in 3T3-L1 cells overexpressing ADAM12 at the cell surface. ADAM12-cyt-expressing cells and control cells were treated with cycloheximide and TNF- to induce apoptosis, and stained with DAPI, and the number of apoptotic cells was counted (A-C). ADAM12-cyt-expressing cells were more prone to apoptosis (A) than were control cells (B). Apoptotic cells (fragmented nuclei) are indicated by arrows. In C, the effect of ß1-activating antibody 9EG7 on TNF--induced apoptosis was examined. Apoptosis in ADAM12-cyt expressing cells (black columns) could be prevented by 9EG7 (*P<0.01), whereas the antibody had no effect on control cells (hatched columns). Fibronectin matrix assembly was altered in ADAM12-cyt-expressing cells (D) compared with control cells (E). Bars, 5 µm (A,B) and 12 µm (D,E).

View larger version (81K): [in a new window]   Fig. 10. Altered extractability of ß1 integrin in Triton X-100 in 3T3-L1 cells overexpressing ADAM12 at the cell surface. Adherent 3T3-L1 ADAM12-cyt cells and control 3T3-L1 cells treated with 0.01% Triton X-100 in DMEM (A,B) for 5 minutes on ice or left untreated (C,D) and subsequently immunostained with polyclonal antibodies to ß1 integrin (A-D). In parallel experiments, adherent 3T3-L1 ADAM12-cyt cells (E) or control cells (F), or normal growing 3T3-L1 cells (G) or confluent day 0 3T3-L1 cells (H) were extracted with 0.5% Triton X-100 in a physiological buffer (CSK) for 5 minutes on ice, released from the plastic substrate with dissociation buffer and immunostained with polyclonal antibodies to ß1 integrin in suspension as described in Materials and Methods. Note that the increase in ß1 integrin solubility, and hence, decreased immunostaining in cells overexpressing ADAM12-cyt (A,E) is similar to that seen in normal confluent day 0 3T3-L1 cells (H) compared with normal growing 3T3-L1 cells (G). Bars, 25 µm (A-D) and 15 µm (E-H).