No abstract

Transforming growth factor-beta (TGF beta) is a local regulator of cell metabolism and growth. TGF beta increases the synthesis of collagen and enhances the deposition of matrix by almost all cells studied to date. The presence of TGF beta in cartilage suggests an important autocrine function, and the present study was designed to examine its influence on the matrix synthesis of chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% fetal bovine serum (FBS), and after 24 h in monolayer culture were treated with TGF beta in identical medium. A 24-h incubation with TGF beta caused a dose-dependent decrease in collagen synthesis (-14%) and increase in noncollagen protein synthesis (+25%), with greater effects in serum-containing medium (-22% and +58%, respectively). Similarly, the stimulation of sulfate incorporation by TGF beta was greater in FBS-containing medium (+140%) than in serum-free medium (+70%). These changes were present by 6 h, were maximal in the 0.3-3.0 ng/ml dose range, and were found to reflect an alteration in extracellular protein synthesis. The enhancement of TGF beta effects by serum was abolished when chondrocytes were plated and exposed to TGF beta in medium containing dialyzed FBS (12-14K membrane). The present study indicates that TGF beta influences the synthesis of matrix components by growth plate chondrocytes. The effects are enhanced by factors present in serum.

Chondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-beta 1 (TGF-beta) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-beta on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-beta in identical medium. A 24 hour incubation with TGF-beta caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 2 1/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-beta. Higher concentrations of TGF-beta were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-beta and serum factors and was abolished when chondrocytes were plated and exposed to TGF-beta in medium containing dialyzed FBS (12-14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-beta-treated and control cultures as the apparent Kms were similar. The present study indicates that TGF-beta increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.

Isolated bone cells in culture contain an enzyme capable of hydrolyzing the phosphate ester of phosphotyrosine. This enzyme, which we have termed phosphotyrosine phosphatase, has not previously been reported in bone. Some of its characteristics include: 1) maximum activity near physiological pH, 2) a Km for substrate of 52 microM, 3) marked inhibition by the phosphate analog vanadate ion, 4) activity correlation with bone cell alkaline phosphatase, and 5) regulation by bone target hormones. Data obtained with vanadate ion support the contention that this enzyme may play a role in the regulation of bone cell growth.

The enzymatic isolation of cells from fetal rat calvaria is most effectively achieved with crude Clostridium histolyticum collagenase. However, this bacterial collagenase damages the cells during the digestion of the tissue. We have used cell density, as measured by isopycnic centrifugation on polysucrose gradients, as an indicator of cell damage. There are at least two enzymes in crude bacterial collagenase capable of damaging the cells in this tissue. One of these is clostripain that has been well characterized. The other cytotoxic enzyme is uncharacterized, and its effects are not evident until the clostripain activity has been inhibited by alpha-tosyl-lysyl chloromethane. The apparent activity of this second enzyme can be inhibited by withholding magnesium from the digestion medium and by increasing the potassium concentration of the digestion medium.