Cell Surface Receptor For Fibronectin Research Articles

The extracellular matrix proteins type I collagen, type III collagen, fibronectin, and laminin 421 stimulate migration of cancer cells.

For metastasis formation, individual cells from a primary tumor must migrate toward other tissues. The aim of this study was to determine if mesenchymal stromal cells (MSCs) from human bone marrow are able to emit signals that induce this migratory activity in cancer cells. We separated the supernatant of MSCs derived from human bone marrow by size-exclusion and ion-exchange chromatography and have subsequently studied the migratory behavior of the prostate cancer cell line PC3 and the breast cancer cell line MDA-MB-231 toward the respective fractions in a transwell migration assay. We identified the extracellular matrix (ECM) proteins type I collagen, type III collagen, fibronectin, and laminin 421 as potential drivers of cancer cell migration. These results could be reproduced using the corresponding isolated or recombinant ECM proteins. Knockdown of the gene encoding beta 1 integrin, an important cell surface receptor for fibronectin, has led to inhibition of cancer cell migration. This supports the hypothesis that beta 1 integrin signaling represents an initial event that leads to metastasis, and that signaling is triggered by binding of integrin heterodimers to ECM molecules. Further characterization of signaling factors and their respective receptors will have implications for anticancer drug development.

Caveolin-1-dependent β1 integrin endocytosis is a critical regulator of fibronectin turnover

beta1 integrins are major cell surface receptors for fibronectin. Some integrins, including beta1 integrins, are known to undergo constitutive endocytosis and recycling. Integrin endocytosis/recycling has been implicated in the regulation of cell migration. However, the mechanisms by which integrin endocytosis/recycling regulates cell migration, and other biological consequences of integrin trafficking are not completely understood. We previously showed that turnover of extracellular matrix (ECM) fibronectin occurs via receptor-mediated endocytosis. Here, we investigate the biological relevance of beta1 integrin endocytosis to fibronectin matrix turnover. First, we demonstrate that beta1 integrins, including alpha5beta1 play an important role in endocytosis and turnover of matrix fibronectin. Second, we show that caveolin-1 constitutively regulates endocytosis of alpha5beta1 integrins, and that alpha5beta1 integrin endocytosis can occur in the absence of fibronectin and fibronectin matrix. We also show that downregulation of caveolin-1 expression by siRNA results in marked reduction of beta1 integrin and fibronectin endocytosis. Hence, caveolin-1-dependent beta1 integrin and fibronectin endocytosis plays a critical role in fibronectin matrix turnover, and may contribute to abnormal ECM remodeling that occurs in fibrotic disorders.

An adhesion variant of the MG-63 osteosarcoma cell line displays an osteoblast-like phenotype.

MG-63 human osteosarcoma cells were selected for attachment and growth in increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp (RGD) sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5 mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. No increase in fibronectin receptor gene copy number was detected by Southern blot analysis. The peptide-resistant MG-63.3A cells look very different from the MG-63 cells and resemble osteocytes. The resistant cells also grow more slowly than MG-63 cells. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells can regulate the amount of this receptor on their surface in response to environmental factors and that this may affect the phenotypic properties of the cell. MG-63.3A cells differ from MG-63 cells in their ability to form a calcified matrix in vitro and in their increased synthesis of type I collagen. The MG-63.3A cells synthesize 50-100-fold less prostaglandin E2, a mediator of bone resorption, than MG-63 cells. There is an overall down-regulation of chondroitin sulphate proteoglycans in MG-63.3A cells. These results are consistent with the hypothesis that such proteoglycans interfere with calcium phosphate deposition and with the observation that chondroitin sulphate is increased in a wide variety of neoplasms but is absent or in small amounts in normal tissue. We conclude that MG-63.3A cells represent a more differentiated cell type with osteoblast-like properties.

Identification of a Domain on the Integrin α5Subunit Implicated in Cell Spreading and Signaling

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.

Up-Regulation of Integrin α5 Expression by Combination of Substance P and Insulin-like Growth Factor-1 in Rabbit Corneal Epithelial Cells

To clarify the mechanisms by which substance P (SP) and insulin-like growth factor-1 (IGF-1) synergistically facilitate corneal epithelial wound healing, we tested the hypothesis that the combination promotes cell attachment to a fibronectin matrix through up-regulation of expression of integrin α5β1, the major cell surface fibronectin receptor in rabbit corneal epithelial cells. Cultured rabbit corneal epithelial cells were treated with SP and/or IGF-1 and then plated on wells coated with fibronectin and bovine serum albumin. After incubation, the number of cells attached to the wells was counted. In a second experiment, reverse transcription-polymerase chain reaction was used to determine the expression of integrin α5 and β1 by cells pretreated with SP and/or IGF-1. The combination of SP and IGF-1 significantly increased the number of cells attached to the fibronectin matrix and the expression of integrin α5. However, attachment to the fibronectin matrix was inhibited by the addition of GRGDSP, a synthetic peptide that mimics fibronectin. Thus, the synergistic enhancing effect of SP and IGF-1 on the attachment of corneal epithelial cells to the fibronectin matrix and on corneal epithelial migration is partly due to the up-regulation of integrin α5 expression in corneal epithelial cells.

Post-translational Modifications of α5β1 Integrin by Glycosaminoglycan Chains: THE α5β1 INTEGRIN IS A FACULTATIVE PROTEOGLYCAN

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.

Control of fibronectin receptor expression by fibronectin: antisense fibronectin RNA downmodulates the induction of fibronectin receptor by transforming growth factor beta1.

The results of our previous studies of mouse embryo fibroblasts showed that fibronectin expression and fibronectin receptor expression are tightly coregulated and that fibronectin modulates expression of its receptor in response to treatment with the differentiation-inducing agent N,N,-dimethylformamide (Varani and Chakrabarty, 1990, J. Cell. Physiol., 143:445-454; Huang et al., 1994, J. Cell. Physiol., 161:470-482). We also found that transforming growth factor beta1 (TGFbeta1) induces a more differentiated phenotype in the epithelium-derived human colon carcinoma cell line Moser and upregulates the expression of both fibronectin and its receptor (Huang and Chakrabarty, 1994, Int. J. Cancer, 57:742-746). By expressing antisense fibronectin RNA in Moser cells, we have downregulated fibronectin mRNA expression and thus blocked the ability of TGFbeta1 to induce fibronectin expression (Huang and Chakrabarty, 1994, J. Biol. Chem., 269:28764-28768). In this study, we examined the effect of antisense fibronectin RNA expression on the induction of fibronectin receptor by TGFbeta1 and tested the hypothesis that the induction of fibronectin expression by TGFbeta1 is required for the induction of fibronectin receptor expression. Blocking fibronectin induction by TGFbeta1 attenuated the ability of TGFbeta1 to upregulate the expression of cell-surface fibronectin receptors, alpha5beta1 integrin expression, and adhesion to extracellular matrix fibronectin. We therefore conclude that induction of fibronectin expression is required for optimal upregulation of fibronectin receptor expression by TGFbeta1.

Up-Regulation of Integrin α5β1 Expression by Interleukin-6 in Rabbit Corneal Epithelial Cells

Interleukin-6 (IL-6) has been shown to promote the attachment of rabbit corneal epithelial cells to fibronectin-coated substratum and ex vivo migration of the cells on the corneal stroma. To examine whether IL-6 promotes cell attachment through up-regulation of expression of integrin α5β1, i.e., the major cell surface fibronectin receptor, we quantified the levels of both α5 and β1 subunit transcripts by reverse transcription-polymerase chain reaction in cultured rabbit corneal epithelial cells pretreated with various concentrations of IL-6. The levels of both α5 and β1 mRNAs were dose-dependently elevated by IL-6, attaining 1.5- and 1.8-fold increases, respectively, at 10 ng/ml. The stimulatory effect of IL-6 was transient; the levels of both subunit mRNAs reached a maximum 1 h after the addition of IL-6 and returned to the basal levels after 6 h. The IL-6-induced up-regulation of integrin α5 and β1 mRNAs was also confirmed by Northern blot analysis. These results indicate that the increased attachment of corneal epithelial cells to fibronectin and enhanced ex vivo migration on corneal stroma by IL-6 is, at least in part, due to the temporal up-regulation of integrin α5β1 expression in corneal epithelial cells.

Growth and metastasis of human breast cancers in athymic nude mice.

To evaluate critically the merit of utilizing a wound model for growing human tumors, a series of increasingly difficult human tumor types were tested for growth at sites of trauma in athymic nude mice. In vitro tumor lines as well as fresh tumors from the breast, colon, rectum, lung, and a metastasis from an unknown primary were intraperitoneally injected into mice subjected to intra-abdominal organ injury. Successful xenografts were obtained from nine of 10 cell lines and 14 of 24 fresh tumors. The latter included five of six (83%) colon cancers, one lung tumor, metastatic tumor of unknown primary, three of four (75%) metastatic breast cancers and four of six (67%) estrogen receptor (ER)-negative breast primary tumors. Six ER-positive breast tumors tested failed to grow in mice without estrogen supplementation. Xenografts from two breast, two colon and the lung cancers formed spontaneous metastases and all xenografts tested were able to yield serial transplants in the surgical wound model. Histologically, all xenografts and their metastases were identical to their respective donor tumors. Transplantability in mice without exogenous estrogen supplementation was linked to the absence of estrogen and progesterone receptors in breast tumors. Transplantability of the cell lines was associated with the expression of cell surface receptors for fibronectin and hyaluronic acid. Receptors for other extracellular matrix components, namely, laminin, vitronectin, collagen, fibrinogen or von Willebrand factor were not associated with transplantability. These results demonstrate that a large proportion of human tumors, including the breast tumors, can be successfully xenografted into athymic mice by providing them with a healing wound environment, and that such xenografts grown at ectopic sites exhibit metastatic ability.

Effect of selenite on cell surface fibronectin receptor.

Incubation of cells with selenite, under conditions in which there is no effect on cell viability, results in a decrease in the rate of their subsequent attachment to extracellular matrix proteins such as fibronectin (1). The attachment was inhibited by a pentapeptide containing the RGD sequence and by antibody against the cellular fibronectin receptor (alpha 5 beta 1 integrin), indicating that it is receptor-mediated. To investigate whether exposure to selenite has an effect on fibronectin receptors, we assayed for their presence on the cell surface by measuring the ability of cells to attach to a surface that had been coated with antibodies to the receptor. Brief exposure of cells to low concentrations of selenite resulted in a significant decrease in their ability to attach to monoclonal antibodies against the alpha 5 or beta 1 subunits of the fibronectin receptor, as well as to polyclonal antibodies against the complete receptor. This indicates that exposure to selenite results in a decrease in receptors that are present at the cell surface. Exposure of the cells to selenate, selenocystine or selenomethionine did not result in a significant decrease in cell surface receptors. Preincubation of the cells with selenite was required for the effect, indicating that selenite does not directly interfere with receptor structure or function.

The effect of sphingosine on the release of fibronectin from human lung fibroblasts

Previous work from our laboratory (Scheidl et al. (1992) Biochim. Biophys. Acta 1135, 295–300) has shown that sphingosine (Sph), a known inhibitor of protein kinase C (PKC) inhibited the release of fibronectin (FN) into the media of fibroblast cells in culture and that this effect was independent of PKC. We now report that the action of Sph was time dependent (maximum inhibition in 2 h), concentration dependent (0.5–10 μM, at which concentration Sph was not toxic), and rapidly reversible after removal of Sph. Incubating with [ 35S]methionine, we found Sph reduced FN release either by inhibition of FN synthesis or FN secretion. To distinguish between these two possibilities, cells were treated with monensin, an inhibitor of FN secretion, which together with Sph showed no additive effect on FN release. Cycloheximide also inhibited FN release, but this inhibition was additive to that with monensin. We concluded that Sph inhibited secretion of FN, not synthesis. Furthermore, intracellular FN declined less in Sph-treated than in untreated control cells. By cell-surface iodination with Na 125I and lactoperoxidase, followed by immunoprecipitation with antibodies to FN and α 5 β 1-integrin, we showed that labeled multimeric and dimeric cell-surface FN declined, whereas integrins remained unchanged upon Sph treatment. This result suggested that though the number of cell-surface receptors for FN was not affected by Sph, their affinity may be reduced. In addition, we found that either pre- or co-treatment for 30 min with Sph caused a robust inhibition of cell adhesion to FN-coated plastic. We conclude that Sph rapidly inhibits FN secretion, lowers cell-surface FN, and greatly reduces cell adhesion to a FN substratum.

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.

Myosin light chain kinase inhibitors ML-7 and ML-9 inhibit mouse lung carcinoma cell attachment to the fibronectin substratum

We studied the effects of various protein kinase inhibitors on the attachment of mouse lung carcinoma 3LL cells to the fibronectin (FN) substratum. Calmodulin antagonists (W-7 and W-13) and myosin light chain kinase inhibitors (ML-7 and ML-9) exhibited the inhibitory effect for the attachment, while inhibitors of protein kinases A and C were ineffective. Since Arg-Gly-Asp-containing hexapeptide blocked the attachment, cell surface FN receptor appeared to be involved in this mechanism. These results support the hypothesis that the cell attachment requires the rearrangement of the cytoskeleton in association with the phosphorylation of myosin light chain which would lead to the clustering of the cell surface FN receptors.

Intracellular events are responsible for the differential expression of fibronectin on the fibroblast surface during chick embyro development

We previously showed that differences in the adhesive behaviour of fibroblasts obtained from 8-day-old (8-day CEF) and 16-day-old chick embryos (16-day CEF) were not due to alterations of cell surface fibronectin receptors. Herein we show that fibronectin (FN) was expressed more rapidly on the 8-day CEF surface (30 min) than on the 16-day CEF surface (60 min). In order to elucidate the mechanism responsible for these differences in the expression of cell surface FN we investigated the biosynthesis and the post-translational modifications of FN in 8- and 16-day CEF. Pulse-chase experiments revealed that FN was processed more slowly to an endo- β- N-acetylglucosaminidase H (endo H)-resistant form in 16-day CEF than in 8-day CEF, whereas the kinetic of FN biosynthesis was similar in both cell populations. This difference was not related to a differential retention of FN in endoplasmic reticulum (ER) as determined after saponin-permeabilization. These results suggested that the rate-limiting step in the transport of FN to the cell surface in 16-day cells occurred between the ER and the medial part of the Golgi apparatus. It seem that the delay in the processing of endo H-resistant N-glycans was sufficient to account for differences between 8- and 16-day CEF in the rate of surface expression of FN and CEF adhesion to a plastic substratum.

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes

Several cell types participate in the formation of vitreoretinal traction membranes in proliferative intraocular disorders. The communication between these cells involves hormones, growth factors, and the interaction with extracellular matrix molecules. We have previously demonstrated a partial colocalisation of two potent mediators of cell attachment, fibronectin and vitronectin, in periretinal membranes from patients with proliferative vitreoretinopathy (PVR). We found a similar pattern of vitronectin and fibronectin deposition in proliferative diabetic retinopathy (PDR) (n = 6). Now we show the expression of the corresponding cell surface receptors, integrins, for fibronectin and vitronectin by proliferating cells in 22 periretinal membranes, including traumatic (n = 8) and idiopathic (n = 8) PVR as well as PDR membranes (n = 6). Integrins are membrane receptors for extracellular matrix macromolecules which are involved in such basic biological phenomena as embryogenesis and metastasis. Future studies on the pathogenesis of vitreoretinal proliferation will have to focus on the initiation, maintenance, and regulation of this intercellular communication network involving attachment proteins and integrins.

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes

Vitronectin and proliferative intraocular disorders. II. Expression of cell surface receptors for fibronectin and vitronectin in periretinal membranes

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.

Internalization of the fibronectin receptor is a constitutive process

Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: 1) after saturation binding of an anti-FnR antibody (PB1) to cells at 4 degrees C, internalization was initiated by warming to 37 degrees C, and then acid/salt elution of membrane-bound ligand was used to quantitate the internalized 125I-PB1; 2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and 3) selective immunoprecipitation was used to separate surface and internalized 125I-labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70-75%) is apparently shed from the cell upon ligation with antibody at 37 degrees C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.

Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.

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.