Autocrine Transforming Growth Factor-β1 and β2 Expression Is Increased by Cell Crowding and Quiescence in Colon Carcinoma Cells

Abstract Although a great deal is known about the cellular effects of exogenous transforming growth factor-β (TGFβ) treatment and the effects of various exogenous agents (including TGF-β's themselves) on TGF-β expression, studies of cellular controls for autocrine TGF-expression and function have been rare. Since exogenous TGF-β treatment blocks progression through the cell cycle, it seemed likely that autocrine TGF-β activity would be induced by growth states in which there was little or no cell division such as confluency or quiescence. Specific TGF-β 1 or β 2 neutralizing antibody treatment of a colon carcinoma cell line designated CBS showed that autocrine TGF-β activity could be demonstrated in quiescent cells but not in preconfluent cells. Studies of kinetics of TGF-β 1 and β 2 mRNA levels during the establishment of quiescence revealed a significant increase of both isoforms in quiescent cells. The quiescent cells also secreted three- to fourfold and four- to fivefold higher levels of total (latent plus active) TGF-β 1 , and β 2 protein in the conditioned media than the confluent cells and preconfluent cells, respectively. There was no detectable active form of either TGF-β isoform in the conditioned media of preconfluent cells, whereas a significant amount of active TGF-β 1 and β 2 was detected in the conditioned media of quiescent cells. Quantitative RNase protection assays were developed to compare the effects of cell crowding vs quiescence on TGF-β expression. TGF-β 1 was primarily induced by quiescence. TGF-β 2 was induced by both quiescence and cell crowding. Increased TGF-β 1 mRNA levels appeared to be exclusively due to an increase in stability, while increased TGF-β 2 mRNA levels were due to increased transcription. This growth state-related induction of TGF-β's was also observed in two other colon carcinoma cell lines. These studies show that TGF-β 1 and β 2 are autocrine-negative factors which can be situationally expressed by cells as a function of their growth state. Autocrine expression of the TGF-β's in this model system appears not to affect exponentially growing cells, but rather to function by maintaining a quiescent state and/or by blocking progression through the cell cycle.