Fig. 4. Adamts3, not Adamts2, has a role in procollagen I processing in the skeleton. (A) Normal craniofacial development, bone structure and minimal tooth involvement in Adamts2^-/- mice. mCT analysis and three-dimensional reconstruction of skulls of Adamts2^-/- mice and wild-type (+/+) littermates do not show differences in overall patterning, size, dimensions or organization of any craniofacial elements. The lower panels show tooth morphology from littermates. Note the poorly defined topography of molars from Adamts2^-/- mice, although the overall morphology is similar. (B) Adamts3, but not Adamts2 or Adamts14, was strongly expressed during tooth development in the dental papilla (DP), and was expressed less strongly in the ameloblasts (arrow). (C) Expression of ADAMTS3 in stably transfected 293 cells. Conditioned medium from cells was analyzed by western blotting with a polyclonal antibody to ADAMTS3. Note that upon treatment with PNGase F, ADAMTS3 migrates faster. Untransfected cells showed no protein. Molecular mass markers are indicated on the right. (D) Rescue of the procollagen I processing defect by ADAMTS2 and ADAMTS3 in dermatosparactic calf skin fibroblasts (DS). DS were plated alone or mixed with an identical number of HEK-293 cells stably transfected with an empty expression vector (Ct), or with the same vector encoding ADAMTS2 (TS2) or ADAMTS3 (TS3). After two days in culture, western blotting of a non-reducing SDS-PAGE gel was used to detect type I collagen. Note that the antibody used detects both 1 and 2 chains, but reacts more strongly with the 1 chain. The identity of collagen polypeptides associated with the cell layer or recovered from the conditioned culture medium is indicated between the two panels. (E) Rescue of the procollagen III processing defect by ADAMTS2 and ADAMTS3 in DS. Samples used in D were also analyzed by western blotting using an anti-collagen III antiserum. The lower amount of collagen synthesized by DS cultured alone, as compared to co-cultures, was repeatedly observed and may be related to the lower level of confluence in the monoculture. Alternatively, HEK-293 cells may secrete factors activating the synthesis of procollagen. Scale bar: 100 µm.