John P. Pennypacker

Inhibition of limb chondrogenesis in vitro by vitamin A: Alterations in cell surface characteristics

Abstract Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.

The influence of an adhesive cell surface protein on chondrogenic expression in vitro.

Abstract Fibronectin is a major glycoprotein associated with fibroblasts and other cells of mesenchymal origin. However, when mesenchyme differentiates into cartilage, fibronectin is no longer synthesized. The significance of the change in fibronectin was further evaluated by culturing chondrocytes in the presence of exogenous fibronectin. Treatment with fibronectin caused the chondrocytes to assume a fibroblastic morphology and also enhanced other fibroblastic properties. These results suggest that decreased fibronectin levels may be required for chondrogenesis to occur normally.

Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A

Abstract Mesenchyme cells derived from limb buds of day 10 mouse embryos were plated out at confluent and sub-confluent cell densities. Cells in confluent cultures multiplied and differentiated into chondrocytes. The addition of vitamin A to the culture medium inhibited both cell proliferation and chondrogenesis. However, cytosine arabinoside, which also inhibited growth, did not block chondrogenesis. This indicates that the inhibition of growth in the vitamin A-treated cultures did not necessarily contribute to the inhibition of chondrogenesis. Cells in sub-confluent cultures multiplied but did not differentiate into chondrocytes. In contrast to confluent cultures, vitamin A did not inhibit growth in sub-confluent cultures. This observation suggests that vitamin A may inhibit growth by causing contact inhibition.

Enhanced cellular fibronectin accumulation in chondrocytes treated with vitamin A

Chick sternal chondrocytes cultured at high cell density lack fibronectin as a surface protein, while vitamin A-treated chondrocytes contain it as the major cell surface protein. We investigated the mechanism of fibronectin accumulation under these conditions. Control chondrocytes synthesized nearly as much fibronectin as vitamin A-treated chondrocytes, but it was secreted primarily into culture medium. Althought the fibronectin of control chondrocytes was of a slightly lower apparent molecular weight than the fibronectin synthesized by the treated cells, it bound as effectively to the cell layer of both normal and treated cells. In contrast, the vitamin A-treated cultures were 2.7 fold more effective in binding fibronectin synthesized by either control or treated cells. Thus in chondrocytes, vitamin A appears to regulate the cellular accumulation of fibronectin by increasing the ability of the cell layer to bind fibronectin rather than by altering its synthesis or its adhesivity for the cell layer.

Altered proteoglycan metabolism in mouse limb mesenchyme cell cultures treated with vitamin A.

Abstract The inhibition of chondrogenesis by vitamin A was examined in mouse limb mesenchyme cultures. Chondrogenesis in control cultures was characterized by increased synthesis of proteoglycans composed predominantly of chondroitin sulfate. The proteoglycans synthesized in vitamin A cultures were smaller than those from cartilage and were found to contain mainly heparan sulfate and dermatan sulfate. No indication of increased proteoglycan degradation resulting from vitamin A treatment was observed. The similarity in size and glycosaminoglycan composition of proteoglycans from control cultures prior to chondrogenesis and vitamin A cultures suggests that vitamin A maintains the cells in a mesenchyme-like state. Vitamin A also stimulated the mannosylation of a specific fraction of glycopeptides.

Brachymorphic mice (bm/bm): A generalized biochemical defect expressed primarily in cartilage

Abstract Homozygous brachymorphic (bm/bm) mice have a disproportionately short stature. Previous studies have shown that the cartilage proteoglycan is undersulfated as a result of decreased 3′-phosphoadenosine 5′-phosphosulfate (PAPS) levels. In the studies reported here, PAPS synthesizing activity was found to be decreased in both skin fibroblasts and prechondrogenic mesenchyme, but sulfation of glycosaminoglycan was normal in those tissues unless glycosaminoglycan synthesis was enhanced by β- d -xyloside. Furthermore, undersulfation was correlated with increased proteoglycan synthesis as the limb mesenchyme cultures underwent chondrogenesis, and sulfation proceeded in an “all or none” manner. These observations demonstrate that the molecular defect in bm/bm mice is not restricted to cartilage, but is manifested there because of the large amount of chondroitin sulfate synthesized.