title: Establishment of porcine pulp-derived cell lines and expression of recombinant dentin sialoprotein and recombinant dentin matrix protein-1.
abstract: The major non-collagenous proteins in dentin have extensive post-translational modifications (PTMs) that appear to be odontoblast-specific, so expression of recombinant dentin proteins in other cell types does not achieve the in vivo pattern of PTMs. We established cell lines from developing porcine dental papillae and used them to express recombinant dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP1). Pulp cells were immortalized with pSV3-neo and clonally selected. Cell lines were characterized by reverse transcruption-polymerase chain reaction (RT-PCR) and assayed for alkaline phosphatase activity and mineralized nodule formation. One of the five cell lines (P4-2) exhibited an odontoblastic phenotype, as determined by expression of tooth-specific markers, response to cytokines, and ability to form mineralized nodules. DSP and DMP1 expression constructs were transiently transfected into various cell lines. DSP, expressed by P4-2 cells, contained chondroitin 6-sulfate, which is a defining modification of the DSP proteoglycan. DMP1 was secreted and cleaved by proteases, even in human kidney 293 cells, which normally do not express DMP1, demonstrating susceptibility to non-specific proteolysis. Both recombinant proteins enhanced P4-2 cell attachment in a dose-dependent manner. We conclude that we have immortalized porcine odontoblast-like cells which express recombinant dentin extracellular matrix components with post-translational modifications that closely resemble those produced in vivo.
authors.affiliation: University of Michigan Dental Research Laboratory, Ann Arbor, MI, USA.
authors.forename: Takanori
authors.initials: T
authors.lastname: Iwata
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
authors.forename: Isao
authors.initials: I
authors.lastname: Ishikawa
authors.forename: Jan C-C
authors.initials: JC
authors.lastname: Hu
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D002113:Calcification, Physiologic; D002448:Cell Adhesion; D002461:Cell Line, Transformed / Q000378:metabolism; D003782:Dental Pulp / Q000166:cytology* / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis*; D010006:Osteoblasts / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins; D013552:Swine; D019098:Telomerase / Q000378:metabolism; D000099311:Dentin Sialophosphoprotein
chemical_list: D000469:Alkaline Phosphatase; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D019098:Telomerase; D000099311:Dentin Sialophosphoprotein; C513309:DMP1 protein, human