Growth signalling through the alpha 5 beta 1 fibronectin receptor.

This study demonstrates that perturbation of the fibronectin receptor (FNR), a member of the integrin family of adhesion receptors, can stimulate growth of non-transformed epithelial cells but not of transformed epithelial cells. Using the non-adherent cell line FA-K562 we demonstrate that growth stimulation via FNR ligands occurs rapidly and independently of any effects on cell adhesion. Low valence FNR ligands such as glycine-arginine-glycine-aspartate-serine (GRGDS) are the most potent stimulators of the cell cycle regulatory kinase cdc2. Partial synchronization and Western blotting studies suggest that GRGDS affects cdc2/cyclin A complexes in cells in S/G2 phase of the cell cycle. These studies suggest that FNR-mediated growth control appears to be a common feature of transformation. These data suggest that the FNR may be physiologically important in growth control, especially in the presence of low valence, proteolytic degradation fragments of FN. Furthermore, escape from FNR-mediated growth control may be a common feature of transformation.

Modulation of epidermal differentiation by epiligrin and integrin alpha 3 beta 1.

We previously reported that integrin alpha 3 beta 1 mediates epidermal intercellular adhesion as well as cell-substrate adhesion. P1B5, an anti-alpha 3 beta 1 specific monoclonal antibody, is a potent in vitro trigger of epidermal cell-cell adhesion and an inhibitor of cell-substrate adhesion. We now show that P1B5 specifically induces the intercellular localization of integrins alpha 2 beta 1 and alpha 3 beta 1, consistent with its role in inducing intercellular adhesion via these two integrins. P1F2, another anti-alpha 3 beta 1 antibody, does not induce either intercellular adhesion or intercellular accumulation of alpha 3 beta 1 and alpha 2 beta 1. Growth of epidermal cells in high calcium, known to induce epidermal differentiation, also induces intercellular accumulation of alpha 3 beta 1 and alpha 2 beta 1 and increased cell-cell adhesion. We therefore asked whether P1B5 treatment induces epidermal differentiation. P1B5 treatment induces changes consistent with epidermal differentiation, including increased involucrin expression, stratification, and production of squames. P1F2 treatment has none of these effects. In vivo, epidermal basal cells are in close contact with the epithelial basement membrane component epiligrin. Growth of keratinocytes on purified epiligrin but not other matrix components specifically reduces involucrin expression by P1B5-treated keratinocytes. These results suggest that integrin alpha 3 beta 1 has a unique role in epidermal differentiation, that the epitope recognized by P1B5 is involved in triggering this differentiation, and that keratinocyte adhesion to epiligrin inhibits alpha 3 beta 1-mediated differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion.

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity-purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.

Fibronectin receptor modulates cyclin-dependent kinase activity.

The high affinity fibronectin receptor (FNR) is expressed by hematopoietic cells, fibroblasts, and proliferating epidermal cells. Expression of this integrin is altered by chemical and viral transformation, suggesting that FNR dysfunction may play a role in growth control. This study demonstrates that exposing FA-K562 cells to glycine-arginine-glycine-aspartate-serine (GRGDS), a peptide ligand of the FNR, specifically stimulates p34/cdc2- and cyclin A-associated kinase activities. This occurs within 2 h of peptide addition. The 110-kDa form of the retinoblastoma protein appears within 3 h of GRGDS addition, consistent with activation of a G1/S kinase. DNA staining profiles demonstrate that GRGDS induces cell cycle progression within 24 h. Increased anchorage-independent growth is subsequently observed in GRGDS-treated FA-K562 cells. The control peptide, GRGES, which cannot bind the FNR, has none of these effects. This demonstrates that an extracellular integrin ligand can regulate cell proliferation. Furthermore, these results suggest that integrins link the extracellular environment and intracellular growth regulators.

Human epidermal keratinocyte expression of sialyl-Lewis X.

Cell-surface oligosaccharides can function as ligands for intercellular adhesion receptors, matrix proteins, and growth factors. We report that human neonatal and adult epidermal keratinocytes (KC) express sialyl Lewis X [s-Le(x); SA alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3R], a ligand for endothelial and platelet selectins. Freshly isolated or cultured KC bind FH6 monoclonal antibody (MoAb), which is specific for s-Le(x)-containing oligosaccharides. The relevant epitope is bona fide s-Le(x), because sialidase treatment of KC suspensions abrogates FH6 binding while generating de novo KC reactivity with anti-Le(x). KC stained in ice-cold suspension display a knobby membrane distribution of s-Le(x) detectable by immunofluorescence microscopy. As others have reported, FH6 appeared not to bind KC in perpendicular skin sections. However, basal KC in intact epidermal sheets exhibited obvious "honeycomb" reactivity with FH6 when stained and viewed en face, suggesting that s-Le(x) in intact epidermis may occur in bands that parallel the major tissue axis. FH6 specifically immunoprecipitated proteins of Mr 34 kd, 44 kd, and 56 kd from [35S]-labeled KC, and anti-Le(x) precipitated similar proteins from sialidase-treated KC. The enzymatic basis for KC s-Le(x) expression was studied by analyzing acceptor specificities and other properties of KC fucosyltransferases. Results indicate that KC express both Lewis- and myeloid-type alpha 1-3fucosyltransferases. KC s-Le(x) could be an important element of the epithelial milieu, because both epithelial cells and immune cells that home to epithelia express s-Le(x) and related structures, and because KC s-Le(x) is well positioned for selectin-mediated platelet binding after trans-cutaneous wounding. The apparent distributions of s-Le(x) in epidermis and on isolated KC are compatible with a functional role for s-Le(x) in these intercellular interactions.

The relationship of serum IL-6 levels to acute graft-versus-host disease and hepatorenal disease after human bone marrow transplantation.

The potential involvement of cytokines in acute graft-versus-host disease led us to analyze interleukin-6 in serial serum sets from 22 allogeneic marrow recipients who developed either grade 3 or 4 GVHD (n = 10), grade 2 GVHD (n = 6), or grade 1 or no diagnosed GVHD (n = 6). A total of 279 serial serum samples taken three times weekly before day 35 were analyzed. Maximum IL-6 levels were greater than 40 U/ml (range, 40-1536 U/ml), 11-40 U/ml, and less than or equal to 10 U/ml for six, eleven, and five patients, respectively. Serum IL-6 peaks were temporally related to onset of GVHD, onset of a syndrome of hepatorenal dysfunction (HRD), or bilateral lung infiltration. Eight of ten patients who developed grade 3 or 4 GVHD overall had IL-6 maxima of greater than 10 U/ml an average of 1.5 +/- 1.8 days before the clinical onset. Fifteen of 17 patients with peak IL-6 levels greater than 10 U/ml developed symptoms of hepatic and renal dysfunction within three days of the peak, while none of five patients with less than or equal to 10 U/ml of Il-6 developed HRD. Regression analysis demonstrated a linkage between the log magnitudes of the serum IL-6 peaks and onset of either GVHD or HRD within three days (P = 0.001). Furthermore, IL-6 peaks tended to precede GVHD onset for the 10 patients whose GVHD onset and IL-6 peak were within three days of each other (P = 0.02). These results, confirmed by both specific bioassay and by IL-6 ELISA, support the idea that acute GVHD in humans involves a cytokine cascade that includes production of IL-6 in addition to the previously reported involvement of tumor necrosis factor alpha and interferon-gamma.

Fibronectin receptor overexpression and loss of transformed phenotype in a stable variant of the K562 cell line.

A variant of the K562 erythroleukemia cell line, FA-K562, was selected by cycles of adhesion to solid-phase plasma fibronectin (FN). FA-K562 expresses fourfold more cell-surface alpha 5 beta 1 fibronectin receptor (FNR) than parental K562. In addition to expected differences in adhesion to FN, other differences between FA-K562 and K562 implicate this FNR in the regulation of cell growth and morphology. FA-K562 proliferates slowly in liquid culture, its cloning efficiency in soft agar is only approximately 10% compared with approximately 85% for parental K562, and it is nontumorigenic in nude mice. The reduced soft agar growth potential of FA-K562 involves FNR function, because either glycine-arginine-glycine-aspartate-serine (GRGDS) or monoclonal anti-alpha 5 antibody in the agar medium increased cloning efficiency of FA-K562 about fivefold. Morphologically, FN-adherent FA-K562 become fibroblastoid in appearance, assemble filamentous actin, and differ from K562 in vimentin staining intensity and pattern. Soluble GRGDS peptide inhibits both FA-K562 adhesion to FN and the associated cytoskeletal changes. These findings link the alpha 5 beta 1 FNR to both the transformed phenotype and morphology of FA-K562.

Evidence for phosphatidylinositol-linked forms of human and avian fibronectin receptors.

Phosphatidylinositol (PI)-linked forms of surface molecules have been hypothesized to mediate the initial stages of cell adhesion or signal transduction. We report evidence for the occurrence of a functional PI-linked subset of cell surface fibronectin receptors (FNR). Treatment of human MG63 osteosarcoma cells or primary chicken embryo fibroblasts (CEF) with PI-specific phospholipase C (PI-PLC) reduced cell surface FNR expression by 30% as detected by immunofluorescence. PI-PLC treatment of cell membranes purified from [35S]methionine-labeled CEF or MG63 cells led to a similar loss of membrane-associated immunoprecipitable FNR from the pelleted membranes, while such treatment led to the appearance of FNR in the supernatant of treated MG63 membranes. Biosynthetic labeling of CEF FNR with [3H]palmitate and [3H]ethanolamine demonstrated the acylation and putative PI linkage of avian FNR subunits. PI-PLC treatment of CEF and MG63 cells also reduced fibronectin-specific adhesion in a short-term in vitro assay, suggesting that the avian and human FNR occur in PI-linked isoforms which appear to contribute to cell adhesion to fibronectin.

Phorbol ester induces increased expression, altered glycosylation, and reduced adhesion of K562 erythroleukemia cell fibronectin receptors.

The human multipotential hematopoietic cell line K562 expresses fibronectin receptor (FNR) subunits of 160 kDa (alpha chain) and 120 kDa (beta chain). Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to reduced binding of K562 to immobilized fibronectin (FN), although treated cells expressed 10-fold more cell surface FNR than untreated cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed this and showed altered electrophoretic mobilities of FNR subunits from TPA-treated cells. TPA treatment affected N-linked glycosylation, as tunicamycin treatment of K562 cells abolished differences in FNR mobility. Sialidase treatment of FNR immunoprecipitates minimized and sialidase treatment of intact cells eliminated these mobility differences between subunits from control and TPA-treated cells. Reduced sialylation of FNR from TPA-treated cells was further demonstrated by chromatography with bead-coupled lectins and by the greater negative charge of untreated K562 FNR subunits in two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis. A relationship between reduced FNR sialylation and reduced FN binding was suggested by adhesion assays of sialidase-treated K562 which showed that desialylation of cell surface FNR was associated with decreased cell adhesion. Thus, TPA treatment reduces the function, increases the expression, and alters the structure of K562 FNR, and these changes appear to involve FNR sialylation.

Can effective birth control be legislated? An analysis of factors that predict birth control utilization.

PIP: 106 low income sexually active women ages 15-30 years were interviewed at a hospital clinic to determine whether the psychosocial characteristics associated with effective use of contraception are susceptible to policy shifts (e.g., changes in the availability of welfare benefits or abortion services). 60% of the women reported always using birth control and 40% reported ineffective use of birth control. Effective use of birth control was positively related to years of education, satisfaction with present birth control method, and marital status. The relative risks associated with education were 50% greater than thos associated with satisfaction and marital status. None of the 4 variables identified as indicators of the sensitivity of contraceptive practice to changes in public assistance or abortion policies (public assistance status, prediction regarding completion of an unplanned pregnancy, assessment of the consequences of motherhood, and abortion history) was predictive of birth control use. These results strongly suggest that the psychosocial characteristics distinguishing effective from ineffective contraceptors are not those that are directly suscpetible to regulatory, legislative, or judicial restrictions. Rather, those concerned with family planning should support outreach programs that attract poor students to school. In addition, family planning programs should aim to work with clients to improve satisfaction with the methods of contraception chosen.^ieng

Pregnancy-resolution decisions. What if abortions were banned?

This study addressed two related questions: (1) what sets of psychosocial characteristics distinguish women who have had an abortion from those who have not? (2) What sets of characteristics distinguish women who, given their present situations, predict they would terminate an unplanned pregnancy from those who predict they would carry such a pregnancy to term? Fifteen variables associated with pregnancy-resolution decisions were tested in multiple logistic regression analyses. Only number of children was strongly (and positively) predictive of having had an abortion. Four variables predicted expectation that an unplanned pregnancy would be terminated. These results suggest that a ban on abortions would affect virtually all women in the study cohort and that the pregnancies affected would be those that occur when the women are least able to care for a (or another) child and when they feel somewhat positive about their potential for self-improvement.