A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically.

We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using G0-synchronized NRK and NIH-3T3 cells, we showed that anchorage-independent growth is regulated by an attachment-dependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae. In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion. Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition. In related studies, we show that TGF-beta 1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta 1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta 1 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-beta 1 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-beta 1 on anchorage-independent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S. Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-beta 1 requirement for G1/S transit while retaining their normal mitogen requirements for proliferation. These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited. The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-beta 1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage-independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence.

TGF-beta is a critical mediator of acute lung injury.

We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.

Cell attachment controls fibronectin and alpha 5 beta 1 integrin levels in fibroblasts. Implications for anchorage-dependent and -independent growth.

We have examined the effect of cell attachment on fibronectin and alpha 5 beta 1 integrin levels in three distinct anchorage-dependent fibroblast cell lines. Analysis of long-term biosynthetically labeled proteins from parallel cultures of adherent and nonadherent cells showed that the steady-state level of extracellular fibronectin is decreased upon loss of attachment. Pulse labeling studies and Northern blot analyses showed that the decrease occurs post-synthetically. A combined approach of surface radioiodination and biosynthetic labeling also demonstrated a selective post-synthetic decrease in cell-surface expression of the alpha 5 beta 1 integrin upon loss of cell attachment. Overall, we estimate that extracellular fibronectin and cell surface alpha 5 beta 1 integrin levels are reduced 5-7-fold in NIH-3T3, 15-20-fold in AKR-2B, and 50-fold in NRK fibroblasts. Finally, we find decreased total (serum- and cell-derived) fibronectin bound to the surface of nonadherent cells consistent with the reduced expression of alpha 5 beta 1 integrin. These results demonstrate a systematic down-regulation of fibronectin and its major receptor upon loss of attachment and suggest a potential mechanism involved in maintenance of the anchorage-dependent phenotype.

Transforming growth factor-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in normal rat kidney fibroblasts. A necessary effect for induction of anchorage-independent growth.

We have previously shown that the expression of alpha(5)beta(1) integrin on the cell surface is dependent upon cell adhesion to the extracellular matrix, and we report here that transforming growth factor-beta (TGF-beta) overcomes this requirement in normal rat kidney (NRK) fibroblasts. Thus, suspended NRK cells treated with TGF-beta show levels of surface alpha(5)beta(1) integrin that are equivalent to those seen in adherent cells. Moreover, several experiments showed that this effect is necessary for the induction of anchorage-independent growth by TGF-beta. First, a kinetic analysis showed that surface expression of alpha(5)beta(1) integrin was restored in TGF-beta-treated NRK cells prior to the induction of anchorage-independent growth. Second, NRK cell mutants that have lost their TGF-beta requirement for surface expression of alpha(5)beta(1) integrin were anchorage-independent in the absence of TGF-beta. Third, an antisense oligonucleotide to the beta(1) integrin subunit or, fourth, stable expression of an alpha(5)-antisense cDNA blocked the ability of TGF-beta to stimulate anchorage-independent growth. Thus, TGF-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in NRK cells, and this effect is necessary for the induction of anchorage-independent growth.

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.