C H Damsky

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.

When suspension cultures of human promyelocytic leukemia cells (line HL60) were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA; 1.6-160 nM), more than 80% of the cells adhered to the plastic substrate within 24 hr. Within the same time period the immature azurophilic granulations typical of HL60 promyelocytic cells disappeared and the nuclear chromatin became more condensed, but the nucleolus was retained. The attached cells stopped dividing and synthesizing DNA. The phenomenon was irreversible and independent of the continuous presence of TPA. Approximately 60% of the untreated cells and of TPA-treated cells bore surface Fc receptors for IgG. Under the experimental conditions used, about 10% of the TPA-treated cells were also able to phagocytize IgG-coated erythrocytes and more than 80% were able to phagocytize latex beads, but untreated controls were unable to do so. Cellular levels of NADase, acid phosphatase, and non-specific esterase were markedly increased after treatment with TPA, whereas little or no increase was seen after treatment with dimethyl sulfoxide (Me2SO), a drug that induces myeloid differentiation of HL60 cells. Peroxidase activity was lower in TPA-treated and Me2SO-treated cells than in HL60 cells. More lysozyme was found in the medium of TPA-treated cells than in the medium of untreated or Me2SO-treated cells. These data indicate that, after treatment with TPA, human promyelocytic leukemia cells can differentiate into cells that have several characteristics of macrophages.

Establishment of the human placenta requires that fetal cytotrophoblast stem cells in anchoring chorionic villi become invasive. These cytotrophoblasts aggregate into cell columns and invade both the uterine interstitium and vasculature, anchoring the fetus to the mother and establishing blood flow to the placenta. Cytotrophoblasts colonizing spiral arterioles replace maternal endothelium as far as the first third of the myometrium. We show here that differentiating cytotrophoblasts transform their adhesion receptor phenotype so as to resemble the endothelial cells they replace. Cytotrophoblasts in cell columns show reduced E-cadherin staining and express VE-(endothelial) cadherin, platelet-endothelial adhesion molecule-1, vascular endothelial adhesion molecule-1, and alpha-4-integrins. Cytotrophoblasts in the uterine interstitium and maternal vasculature continue to express these receptors, and, like endothelial cells during angiogenesis, also stain for alphaVbeta3. In functional studies, alphaVbeta3 and VE-cadherin enhance, while E-cadherin restrains, cytotrophoblast invasiveness. Cytotrophoblasts expressing alpha4 integrins bound immobilized VCAM-1 in vitro, suggesting that this receptor-pair could mediate cytotrophoblast-endothelium or cytotrophoblast-cytotrophoblast interactions in vivo, during endovascular invasion. In the pregnancy disorder preeclampsia, in which endovascular invasion remains superficial, cytotrophoblasts fail to express most of these endothelial markers (Zhou et al., 1997. J. Clin. Invest. 99:2152-2164.), suggesting that this adhesion phenotype switch is required for successful endovascular invasion and normal placentation.

In human pregnancy, placental cytotrophoblasts that invade the uterus downregulate the expression of adhesion receptors that are characteristic of their epithelial origin, and upregulate the expression of adhesion receptors that are expressed by vascular cells. We suggest that this transformation could be critical to endovascular invasion, the process whereby cytotrophoblasts invade the uterine spiral arterioles and line their walls (Zhou et al. J. Clin. Invest. 1997. 99: 2139-2151.). To better understand the in vivo significance of these findings, we tested the hypothesis that in preeclampsia, an important disease of pregnancy in which endovascular invasion is abrogated, cytotrophoblasts fail to adopt a vascular adhesion phenotype. In experiments described here we stained placental bed biopsy specimens from age-matched control pregnancies and from those complicated by preeclampsia with antibodies that recognize adhesion molecules that are normally modulated during this transformation. In preeclampsia, differentiating/invading cytotrophoblasts fail to express properly many of these molecules, including integrin, cadherin, and Ig superfamily members. These results suggest that preeclampsia is associated with failure of cytotrophoblasts to mimic a vascular adhesion phenotype. The functional consequences of this abnormality are unknown, but are likely to affect negatively cytotrophoblast endovascular invasion and uterine arteriole remodeling, thereby compromising blood flow to the maternal-fetal interface.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.