Evidence supporting the role of a proteinaceous, loosely bound extracellular molecule in the cell density signaling...

Normal cells in culture respond to cell density by altering their proliferation rates and their pattern of protein expression. Primary avian tendon (PAT) cells are a case in point where procollagen production increases approximately 10-fold at high cell density while proliferation almost ceases. In an earlier report focusing on the cell density regulation of procollagen expression, the signaling mechanism communicating the presence of other cells was shown to have the characteristics of a loosely bound component of the cell layer. Extending these studies to the cell density regulation of proliferation, the cell density signal (CDS) was again shown to be altered by medium agitation, stimulating cell division. Agitation, however, was only disruptive to cell signaling when there was a high ratio of medium to cells. When sufficient cells were present, agitation was less effective. Therefore, the CDS controlling procollagen production and the CDS controlling the inhibition of growth seemed to be linked because the signaling mechanism is disrupted in a parallel manner by agitation. However, the proliferative response of PAT cells is more complex in that there is also a positive influence at moderate cell density (> 2 x 10(4) cells/cm2) on the rate of cell division. As a consequence, PAT cells would not proliferate into an area of low cell density, but within the same dish would rapidly fill an area of moderate density. PAT cells were capable of filling a gap between high cell density areas if the gap was less than 2 mm. Medium agitation also affected cells at low cell density in a different manner. It was inhibitory if all the cells were at low cell density but it was stimulatory if the cells at low cell density were in close proximity to cells at high cell density. In addition, medium conditioned by agitation over cells at a high cell density would stimulate cells at low cell density to divide and grow out into low cell density regions. Using the growth-promoting activity of the conditioned medium as an assay, this component of the CDS was shown to have unique characteristics: heat, pH, dithiothreitol (DTT) stable; tris ion and protease sensitive. By gel exclusion chromatography it was larger than 100 kDa. But after DTT treatment its mobility shifted to < 30 kDa while retaining activity.