Dmp1-deficient Mice Display Severe Defects in Cartilage Formation Responsible for a Chondrodysplasia-like Phenotype

Abstract

Understanding the molecular mechanisms by which cartilage formation is regulated is essential toward understanding the physiology of both embryonic bone development and postnatal bone growth. Although much is known about growth factor signaling in cartilage formation, the regulatory role of noncollagenous matrix proteins in this process are still largely unknown. In the present studies, we present evidence for a critical role of DMP1 (dentin matrix protein 1) in postnatal chondrogenesis. The Dmp1 gene was originally identified from a rat incisor cDNA library and has been shown to play an important role in late stage dentinogenesis. Whereas no apparent abnormalities were observed in prenatal bone development, Dmp1-deficient (Dmp1 ؊/؊ ) mice unexpectedly develop a severe defect in cartilage formation during postnatal chondrogenesis. Vertebrae and long bones in Dmp1-deficient (Dmp1 ؊/؊ ) mice are shorter and wider with delayed and malformed secondary ossification centers and an irregular and highly expanded growth plate, results of both a highly expanded proliferation and a highly expanded hypertrophic zone creating a phenotype resembling dwarfism with chondrodysplasia. This phenotype appears to be due to increased cell proliferation in the proliferating zone and reduced apoptosis in the hypertrophic zone. In addition, blood vessel invasion is impaired in the epiphyses of Dmp1 ؊/؊ mice. These findings show that DMP1 is essential for normal postnatal chondrogenesis and subsequent osteogenesis.

Identification of Full-Length Dentin Matrix Protein 1 in Dentin and Bone

“…This purported biological function is supported by observations that MC3T3-E1 cells overexpressing DMP1 demonstrate an earlier onset of mineralization and the formation of a significantly larger size of the induced mineralized nodules compared to nontransfected control cells [4]. Findings from Dmp1 knockout mouse experiments and gene mutation studies on human osteomalacia strengthen the conclusion that DMP1 plays an important role in bone and dentin mineralization [5][6][7]. In addition to its direct role in biomineralization, studies indicated that DMP1 may regulate osteoblast-specific and/or odontoblast-specific genes [8,9].…”

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

“…These concentrations were selected based on pilot experiments that determined saturated binding. A previous study had shown that addition of nonspecific IgG at even higher titers did not affect 45 Ca accumulation. The antibody was either added acutely on day 9, 11, or 14, or continuously with each media change starting at each of these days.…”

“…Cell culture dishes (Falcon) were from Becton Dickinson (Franklin Lakes, NJ). 45 Calcium was from Perkin/Elmer (Boston, MA). Trypsin-EDTA was from CELLGRO (Herndon, VA) and Nitex filters from Tetko Inc (Ardsley, NY).…”

“…Any concentration or addition time that caused necrosis or apoptosis greater than that seen in mineralizing controls was not included in the detailed studies. Concentrations that caused maximal changes in 45 Ca uptake (see below) were selected for evaluation. After concentrations were selected, the inhibitors or the carriers they were dissolved in were added either once at day 7, 9, 11, 14, or 16 (for 2 hrs or 2 days), or continuously (with each change of media) from these time points until day 21-22.…”

“…The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45 Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.…”