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First published online 13 February 2007
doi: 10.1242/jcs.03388

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Dual effects of the membrane-anchored MMP regulator RECK on chondrogenic differentiation of ATDC5 cells

Shunya Kondo^1,2, Chisa Shukunami², Yoko Morioka¹, Naoya Matsumoto^1,3, Rei Takahashi⁴, Junseo Oh^1,*, Tadao Atsumi⁵, Akihiro Umezawa⁶, Akira Kudo⁷, Hitoshi Kitayama¹, Yuji Hiraki² and Makoto Noda^1,

¹ Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
² Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
³ Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
⁴ Department of Pathology and Tumor Biology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
⁵ Antibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
⁶ Department of Reproductive Biology and Pathology, National Research Institute for Child and Health Development, Okura, Setagaya, 157-8535 Tokyo, Japan
⁷ Department of Biological Information, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama, 226-8501, Japan

View larger version (37K): [in this window] [in a new window]   Fig. 1. Abundant expression of Reck in cartilage. (A) Mouse embryo multiple tissue northern blot (Clontech) was hybridized sequentially with cDNA probes for Reck and Gapdh (loading control). Each lane contained 2 µg of poly(A)+ RNA from embryos at the indicated stage (days after gestation). (B-G) Detection of Reck mRNA in mouse embryos by in situ hybridization. Sagittal sections of an E13.5 embryo were hybridized with Reck antisense (B) or sense (C) riboprobe. Magnified views of the boxed areas in B are shown in D and E (*), respectively. The areas containing ribs (F) and a femur (G) in a section of E16.5 embryo hybridized with Reck anti-sense probe are also shown. Northern blot data (not shown) indicated that the apparently strong signals in the liver (labeled `L' in panel B and F) are non-specific. Bars, 1.28 mm in B and C; 200 µm in D and E; 320 µm in F and G. (H-J) Detection of Reck mRNA in cultured cells by RNA blot hybridization. Total RNA extracted from the following cultured cells was analyzed by RNA blot hybridization using the Reck cDNA probe. (H) Lane 1: H4-1 (a primary culture derived from human bone marrow containing the cells capable of differentiating into chondrocytes) (Imabayashi et al., 2003; Mori et al., 2005). Lane 2: DEC (chondrocytes obtained from human cartilage) (Imabayashi et al., 2003). Lane 3: the human fibrosarcoma cell line HT1080-transfected with control vector (negative control). Lane 4: HT1080 transfected with RECK expression vector (positive control). (I) Lanes 1 and 2: longer exposure of lane 1 and 2 in H, (under the same conditions as lanes 36). Lane 3: KUM9 (multipotential progenitor cells derived from mouse bone marrow and capable of differentiating into osteocytes, adipocytes, myocytes and neurons) (Kohyama et al., 2001). Lane 4: KUSA-A1 (osteoblasts derived from mouse bone marrow) (Kohyama et al., 2001). Lanes 5 and 6: MDBM (osteoclast progenitor cells derived from mouse bone marrow) and RANKL-treated MDBM (mature osteoclasts), respectively (Takeshita et al., 2000). The amount of total RNA loaded was 10 µg for lanes 1-4 and 4 µg for lanes 5-6. Patterns of ribosomal RNA bands are also shown (bottom panels). (J) Relative intensity of the bands on the blot shown in I. The band intensity determined by densitometry and normalized against the RNA amount is presented as a bar graph for direct comparison. (K) Temporal expression pattern of Reck mRNA in ATDC5 cells during chondrogenic differentiation in vitro. Total RNA (20 µg) from ATDC5 cells that had been allowed to differentiate in culture for the indicated times (days) was subjected to RNA blot hybridization using a Reck cDNA probe (top panel). The same blot was re-probed with a type II collagen cDNA (middle panel; Col-II) to monitor chondrogenic differentiation.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Expression patterns of MMP, collagens and RECK during cartilaginous nodule formation by ATDC5 cells. (A) Temporal expression patterns of Mmps and Reck in differentiating ATDC5 cells. Total RNA (20 µg) extracted from ATDC5 cells that had been incubated for the indicated times was analyzed by RNA blot hybridization using indicated probes. (B) Collagenase activity expressed by differentiating ATDC5 cells. ATDC5 cells were incubated for the indicated times and then overlaid with semi-solid medium containing reconstituted type I collagen and DQ collagen. After an additional 24 hours incubation, morphology (DIC) and collagenolytic activity (green DQ-fluorescence) were recorded with a confocal microscope. Bars, 100 µm in the day-5 and day-10 panels; 200 µm in the day-24 panel. Arrowheads indicate the rim of a large nodule. (C) Sensitivity of the DQ-fluorescent signals to an MMP inhibitor GM6001. The experimental conditions were the same as in B, day 24 except that the cells were exposed to GM6001 (100 µM; bottom panels) or an inactive analogue (100 µM; control) for the last 36 hours (i.e. 12 hours before overlay plus 24 hours after overlay). Bars, 100 µm. (D) Temporal expression pattern of type I collagen mRNA in differentiating ATDC5 cells. The same blot used in A was re-probed with a type I collagen cDNA. (E and F) Localization of type I and type II collagen at cartilaginous nodules formed by ATDC5 cells. ATDC5 cells incubated for 15 days were fixed and stained with anti-RECK (red) plus anti-type I collagen (green; E) or anti-RECK (red) plus anti-type II collagen (green; F). Reconstituted z-axis images along two cutting lines (a,b) are shown in the top and right panels, respectively. Bars, 100 µm in E,F.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effects of forced RECK expression on nodule formation by ATDC5 cells. (A) ATDC5 cells were stably transfected with a mammalian expression vector pCXN2 (V) (Niwa et al., 1991) or the vector expressing human RECK (R). Expression of RECK protein (top panel) and RECK mRNA (middle panel) in the pooled transfectants harvested under the proliferative conditions was analyzed by immunoblot assay (30 µg protein per lane; top panel) and RNA blot hybridization (10 µg total RNA per lane; middle and bottom panels), respectively. Relative band intensity is shown under each lane. (B) Growth curves of the transfectants. The plating density at day 0 was 3x104 cells per 35-mm dish. The data are average values from duplicate dishes. (C) Phase-contrast micrographs of the vector-transfected cells (left panels) and RECK-transfected cells (right panels) incubated for the indicated times. Bars, 300 µm. Arrows indicate compact nodules. (D) Number of foci (areas of cell condensation) formed by the vector-transfected cells (hatched bars) and RECK-transfected cells (white bars) after incubation for the indicated times. The data are given as mean ± s.e.m. of quadrate dishes. Consistent results were obtained in two separate experiments using the same set of transfectants and in another experiment using retroviral vectors for gene transfer.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Effects of forced expression of RECK or MMP on chondrogenic differentiation of ATDC5 cells. (A) The cells transfected with the control vector (left panels) or the vector expressing human RECK (right panels) were incubated for the indicated times and then stained with Alcian Blue. Bars, 2 mm. (B) Expression of chondrogenic differentiation markers in the transfectants. Total RNA (20 µg) extracted from the transfectants at the indicated times (bottom labels) was analyzed by RNA blot hybridization using the indicated probes (top labels). (C) Effects of forced RECK expression on the level of secreted gelatinases. Conditioned media (7 hours conditioned) prepared from vector-(V) or RECK-transfected ATDC5 cells (R) at the indicated times were analyzed by gelatin zymography. The positions of the pro-MMP9, pro-MMP2, and intermediate-MMP2 bands are indicated. The amounts of samples applied were adjusted between V and R so as to represent equal cell number. Day 7 samples were five times more concentrated than day 3 samples on a per-cell basis. (D) Effects of forced expression of MMPs on cellular condensation of ATDC5 cells. ATDC5 cells were stably transfected with a mammalian expression vector (pcDNA3.1/Hygro(+); Invitrogen) or the vector containing mouse Mmp2, mouse Mmp9, or human MT1-MMP cDNA. Phase-contrast micrographs of vector- or MMP-transfected ATDC5 cells incubated for 5 days are shown. Expression of MMP in each transfectant was confirmed by northern blot hybridization and gelatin zymography. Note the accelerated cellular condensation in Mmp9-transfected cells and MT1-MMP-transfected cells. Bars, 100 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Effects of Reck gene knockdown on cartilaginous nodule formation by ATDC5 cells. (A) ATDC5 cells were stably transfected with the control vector (V) or the vector expressing shRNA against Reck (S). Expression of RECK in these cells were analyzed by immunoblot assay (top panel) and RNA blot hybridization (middle panel). Total proteins (30 µg) harvested on day 7 and total RNA (20 µg) harvested on day 4 were used. The number under each lane indicates relative band intensity. (B) Phase-contrast micrographs (top 2 rows) and Alcian blue-stained foci (third row) of the vector-transfected cells (left panels) or RECK-siRNA-transfected cells (right panels) incubated for the indicated times. Bars, 100 µm (Day 12); 250 µm (Day 15) and 400 µm (Day 24). (C) The number and morphology of foci. After incubation for the indicated times, the number of all visible foci (Total foci) and the number of highly refractile foci (e.g. in B, Vector Day 15, arrow), which represent ECM-rich cartilaginous nodules (Shukunami et al., 1998), were scored under a microscope. Values are mean ± s.e.m. from quadruple dishes. (D) Immunofluorescent staining of foci at day 15. The cells were doubly stained with anti-type II collagen (green) and anti-RECK (red) antibodies. Morphology (DIC), type II collagen signals (Col-II), and RECK signals (RECK) around typical foci were recorded under the same microscopic conditions. Bars, 100 µm. The findings were consistent between two separate experiments using independently derived transfectants.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Possible roles of RECK and MMPs during chondrogenic differentiation. (A) Schematic representation of the temporal changes in the amounts of mRNA encoding MMP (representing MMP2, MMP9 and MT1-MMP; red line), RECK (green line), and type II collagen (blue line) during ATDC5 differentiation. (B) A working hypothesis: elevated MMP activity is required for cellular condensation in the early phase and nodule expansion in the later phase in ATDC5 differentiation. Elevated RECK expression is required for ECM accumulation inside the nodules in the later phase. Constitutive RECK expression suppresses cellular condensation. RECK knockdown suppresses ECM accumulation and hence nodule maturation.