In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix

Abstract:

In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [³H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [³H]-d-glucosamine macromolecules synthesized, [³H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% ± 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.