Regulation of cell adhesion receptors by transforming growth factor ...

Vol. 264, No. 1, Issue of January 5,pp: 389-392,1989 Prrnted in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Cell Adhesion Receptors by Transforming Growth Factor-@ REGULATION OF VITRONECTIN RECEPTOR AND LFA-1* (Received for publication, August 17, 1988)

Ronald A. IgnotzS,Jyrki HeinoQ,and Joan Massague From the Department of Biochemistry, University of Massachusetts Medical School, Worcester,Massachusetts 01655

We have examined the ability of transforming growth factor-Bl (TGF-81) to regulate the expression of members of the aj32 and aj33 families of integrins. TGF-81 elevates the expression of vitronectin receptors (a& integrin) in all cellsexamined including WI38 human lung fibroblasts, 3T3-Ll mouse fibroblasts, and MG-63 human osteogenic sarcoma cells. TGF-Bl action increases the level of mRNA and the synthesis of vitronectin receptor subunits with t l D of 3-4 h and 6 h, respectively. TGF-j31 up-regulates expression of the intercellular adhesion receptor, LFA-1 ( a d 2 ) ,in THP-1 human monocytic leukemia cells by increasing the synthesis of aL subunit butnot subunit. The increase in (YLsynthesis and assembly into LFA-1 complexes induced by TGF-81 occurs in parallel with elevated fibronectin receptor synthesis in THP-1 cells. These responses to TGF-81 are lost upon phorbolesterinduced differentiation of THP-1 cells into the macrophage phenotype. The results suggest a role of TGF-j3 in the regulation of cell-matrix interactions mediated by vitronectin receptors and cell-cell interactions mediated by LFA-1 in the immune system.

identical to thatof the vitronectin receptor (10). The third family of integrins is defined by a common p2 subunitand includes three members,LFA-1, Mac-1,and p150,95 (11,12). These integrins are found on the surface of leukocytes and playa broad role in mediating cell-to-cell adhesion in many aspects of the immune response. LFA-1 is expressed on thesurface of most lymphocytes, actsas a receptorfor intercellularadhesion molecule-1 (13),and enhances helper and cytolytic lymphocyte functions as well as antigen-independent adhesion of lymphocytes to othercells (14). Mac-1 and p150,95 are expressed by monocytes and macrophages, bind the C3bi component of complement (15), and participate in adhesion and homing of these cells to endothelial cells a t inflammatory sites (16). The aP1 integrins have been described as targets for regulation by hormonally active polypeptides involved in tissue generation, morphogenesis, and repair (17, 18). A prototype of this class of factors is the transforming growth factors-p (TGF-P).’ The ability of TGFs-(3 to regulate the expression of multiple members of the a@,integrin family hasbeen examined in detail in mesenchymal and epithelial cell types whose morphology, proliferation, or differentiation are controlled by thesefactors(19).Thevitronectinreceptoris functionally homologous to other matrix adhesion receptors whose expression is regulated by TGFs-P, and LFA-1 and related integrins participate in immune cell functions that are potently suppressed by TGFs-j3 (20, 21). Based on this evidence, we reasoned that the expression of vitronectin receptor and LFA-1 might also be regulated by TGF-P. The studies described in the present reportconfirm this possibility.

The integrin gene superfamily of cell adhesion receptors plays a major role in the mediation of adhesive interactions between cellsand the environment. Three families of integrins exist that are individually defined by the type of P subunit a@dimer (1, 2). The aPl integrins present in the integrin function as extracellular matrix receptors with bindingspecificity for fibronectin, collagen, laminin, and other proteins. Their binding specificity is dictated by the type of a subunit EXPERIMENTALPROCEDURES in the a& complex. Antibodies-Antiserum against PI integrin subunit was raised in Asecondfamily of integrinsthatbindtoextracellular rabbits using affinity-purified human placenta fibronectin receptors matrix and plasma proteinsincludes the vitronectin receptor as the antigen (19). Antiserum raised against human platelet IIIa and the platelet gpIIb/IIIa complex. Vitronectin is a promi- protein (p3 integrin subunit) was provided by Dr. R. McEver (Oklanent plasmaglycoprotein that is important for the attachment homa Medical Research Foundation, Oklahoma City, OK). Antisera and spreading of cells in culture and presumably in vivo (3). against human P 2 and O(L subunits were generously provided by Dr. The vitronectin receptor,or a& integrin, has beenidentified T. Springer (Harvard Medical School, Boston, MA). TGF-&Bovine TGF-P1 was prepared from demineralized bovine in endothelial, osteogenic, and fibroblastic cell types (4-7). bone according to Seyedin et al. (22). The platelet integrin gpIIb/IIIa mediates binding to fibrinoCell Labeling and Immunoprecipitation-Cell lines were obtained gen, von Willebrand factor, and fibronectin (8,9). This plate-from the American Type Culture Collection. The cultures were mainlet receptor, also known as a I I & integrin, has a p subunit tained according to the supplier’s recommendations. Subconfluent * This work was supported by National Institutes of Health Grant CA39240 (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Supported by a postdoctoral fellowship from the National Institutes of Health. § Supported by a postdoctoral fellowship from the Academy of Finland.

monolayer cultures, or cell suspensions (THP-1 cells), were treated with or without TGF-Pl in minimal essential medium containing 0.1% calf serum (or serum-free RPMI 1640 for THP-1 cells) for a specified time after which the medium was replaced with cysteinefree, serum-free minimum essential medium containing 20 pCi/ml of [35S]cysteine (-1,000 pCi/mmol, DuPont-New England Nuclear). After a 3-h labeling period, the cells were detached by scraping and/ or recovered by centrifugation at 2,000 X g and 4 “C for 5 min. The

389

The abbreviation used is: TGF-8, transforming growth factor-&

TGF-p Regulates p2 and p3 Integrins

390

cell pellets were solubilized in a buffer consisting of 100 mM n-octyl8-D-glucopyranoside, 10 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 mM CaCI2, 1 mM M&12, and 1 mM phenylmethylsulfonyl fluoride. After 10 min on ice, the insoluble material was removed by centrifugation a t 13,000 X g for 3 min a t 4 "C. The supernatants were adjusted to 0.5% Triton X-100 and 0.5 mg/ml bovine serum albumin and precleared with 10 pl of protein A-Sepharose heads for 30 min a t 4 "C. The precleared supernatants were then treated with the appropriate antisera for 2 h. Immune complexes were recovered with protein ASepharose and washed four times with a buffer consisting of 1% Triton X-100, 1 mg/ml bovine serum albumin, 150 mM NaCI, 10 mM Tris-HCI, pH 7.4, and once with 0.5 M NaCI, 1 mMCaC12, 1 mM MgCI,, 10 mM Tris-HCI, p H 7.4. The immunoprecipitates were then analyzed by electrophoresis on 7% polyacrylamide-sodium dodecyl sulfate gels and fluorography, with Enlightening (DuPont-New England Nuclear) as thefluorographic agent. mRNA Analysis-Northern hybridization analysis for vitronectin receptor a subunits was done using total cellular RNA prepared by the guanidine isothiocynate, CsCl centrifugation procedure (23). The RNA was fractionated on 1%agarose gels containing formaldehyde, transferred onto nitrocellulose, and hybridized to probes labeled with a2P using the Multiprime DNA labeling system (Amersham Corp.). The blots were probed with either a cDNA clone for the vitronectin receptor CY subunit ( 4 ) (a gift of E. Ruoslahti, LaJolla CancerResearch Foundation, California), or a glyceraldehyde-3-phosphate dehydrogenase cDNA clone (24).

reduction, this product was identified as the 8 3 integrin subunit. Several other biosynthetically labeled products were present in the immunoprecipitatedsamples. Some of them were high molecular weightmaterial that also appeared with preimmune serum andyielded several lower molecular weightproducts after reduction. However, a labeled species of 135 kDa (nonreduced) precipitated only with the immune serum; and given its molecular mass (4-7), it probably corresponds to the vitronectin receptor av subunit coprecipitated as part of the vitronectin receptor complex (see below). Treatment of WI-38 cells with TGF-Dl increased approximately 3-fold the amount of labeled 8 3 subunit (Fig. lA). A similar response to TGF-81 was observed in other cell lines examined, including MG-63 human osteosarcoma cells and 3T3-Ll mouse fibroblasts (Fig. 1B). Thus, TGF-B1 action increased the rate of synthesis of vitronectinreceptor 8 3 subunits in the threecell lines examined. avsubunit The labeling of the putative vitronectin receptor also increased in response to TGF-B1. Precipitation of the av subunit using this antibody preparation was due to coprecipitation as a complex with p3 subunit. We sought direct evidence for an effect of TGF-B1 on avsubunit expression. Since an anti-a, subunit antibody was not available for this study, RESULTS AND DISCUSSION the level of avsubunit mRNA present in WI-38 and MG-63 We investigated first the effect of TGF-Pl on the rate of cells was determined after treatment of cells with TGF-Dl. synthesis of vitronectin receptor(a& integrin) in human and TGF-Pl treatment did increase the steady-state level of av murine cell lines. The antibody preparation used to immu- subunit mRNA in both cell lines toa level 3- and 7-fold higher noprecipitate biosyntheticallylabeled vitronectinreceptor than basal, respectively (Fig. 2). The effect of TGF-Dl on the had been raised against human platelet gpIIIa protein (25) level of av mRNA was half-maximal after 4 h and reached a which has the same amino acid sequence as the vitronectin maximum after 8-9 h, as determineddensitometrically. This receptor p3 subunit (10). A radiolabeled product of 90 kDa effect of TGF-B1 was selective as indicated by the constant (nonreduced) was specifically precipitated from WI-38 fibro- level of glyceraldehyde-3-phosphatedehydrogenasemRNA blasts using thispolyclonal antibody (Fig. lA). Under reducused as a control. ing conditions, this labeled product migrated in electrophoThe kinetics of the TGF-B1 effect on vitronectin receptor resis gels as a115-kDaspecies (Fig. LA). Based on these synthesis in WI-38 fibroblasts matched well the kinetics of molecular weight values and the characteristic shift upon the effect at themRNA level. The rate of synthesis of vitronectin receptor was elevated half-maximally and maximally A 6 h and between 9-12 h after TGF-Dl addition, respectively puli3T3-Ll (Fig. 3). The ratio between [35S]cysteine-labeled av subunit -+ and B3 subunit precipitated at all time points was uniform. Based on a 1:3 ratio between the cysteine content of av and 200BB subunits (4, lo), theresultsareconsistentwith a 1:l stoichiometry of both subunits in the receptor complex. 116 The components of the ab2 family of integrins, LFA-1, $94-3 cp3 J!L 941 "B3 Mac-1, and p150,95, are expressed on the surface of monocytes x and macrophages (12). T o examine thepossibility that mem68 bers of this family of integrins might be regulated by TGF68 B1, we examined various cell lines that express these cell adhesion receptors as well as fibronectin receptor, a known I

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FIG. 1. TGF-8 increases synthesis of vitronectin receptor 8 subunit. A, subconfluent cultures of human lung WI-38 fibroblasts were treated with medium alone or containing 250 pM TGF-81 for 9 h followed by a 3-h pulse labeling with 20 pCi/ml [3sSS]cysteine,again with or without TGF-81.After the labeling period, the cultures were harvested andsolubilized. Detergent-soluble cell extracts were treated either with a control sera or the anti-gpIIIa antisera. Immunecomplexes were collected by binding to protein A-Sepharose, heated in the presence (+D7'7') orabsence (-DT7') of dithiothreitol,and subjected to electrophoresis and fluorography. B, cultures of human osteosarcoma MG-63 cell and mouse 3T3-Ll cells were treated as described above. Extracts were prepared, immunoprecipitates made and subjected to electrophoresis andfluorography. In thefluorograms shown, solid arrows indicate the position of the 83 subunit, and the subunit. open arrows indicate the putativeCY,

MG-63 0 3 6 912

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FIG. 2. TGF-8 causes an increase in a, mRNA levels. Cultures of WI-38 and MG-63cells were treated with 250 p~ TGF-8 for the indicated periods of time. Total cellular RNA was prepared and 20 pg was subjected to electrophoresis in 1%agarose and formaldehyde gels followed by Northern blot analysis to detect mRNA for the a subunit ( a v )of the vitronectin receptor. A 7-kilobase hand was detectedwhentheblots wereprobed for vitronectin receptor a, subunit mRNA. Thesameblots were alsoprobedwithacDNA corresponding to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Shown are the regions of the autoradiographs corresponding to the hybrid-labeled mRNAs.

TGF-p Regulates pz and & Integrins

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tated as part of an integrin receptor complex with subunit. This a subunit might be asbecause it was not recognized by " " 0 antibodies against the other known a subunits of the a/31 family of integrins (19). THP-1 cellsdidexpress small 200 level was not altered amounts of a.,integrin subunits, but their by TGF-/31 action (not shown). The effect of TGF-/31 was characterized bya 2-fold increase in total (precursor plus mature) PI subunit level and a larger (4.5-fold) increase in the 116 level of the coprecipitatinga subunit. When similar experimentswere carried out using antibody 8 94Y against the p2 integrin subunit, TGF-/31 did not increase the -p 3 level of newly synthesized p2 subunit in THP-1 monocytes but increased the level of a 180-kDa coprecipitating product (Fig. 4). The molecular weight and coprecipitation of this 68product withp2 subunit raised the possibility that this product ) in the LFA-1 complex. might be the a subunit ( a ~present This possibilitywasconfirmed by immunoprecipitation of extracts from labeled cells with an anti-aL monoclonal antibody (Fig. 5). A similar effect of TGF-Pl was seen in experiFIG. 3. Kinetics of vitronectin receptor induction. Cultures ments with another human monocytic cell line, U937 (not of WI-38 fibroblasts were treated with 250 PM TGF-01 for the shown). indicated periods of time. The cultures were given [3sS]cysteine for Phorbol myristate acetate-inducedmacrophage differentiathe final 3 h of TGF-B1treatment as in Fig. 1. The cells were tion of THP-1 cells led to changes in the biosynthesis of harvested, detergent-solubilized, andtheextractsimmunoprecipiBl subunit and its integrins. The molecular weight of the tated with anti-gpIIIa antisera. The immunoprecipitates were sepawas different frommonocytes probrated on polyacrylamide gels (nonreduced) and subjected to fluorog- precursor in macrophages ably due todifferences in glycosylation in the two phenotypes raphy. macro(Fig. 4). More importantly, after differentiation to w e Mxrqlhage W e MxrophaEp phages THP-1 cells lost the ability to respond to TGF-Bl 12 24 12 24 12 24 12 24 Hours " " " " - + - + - + - + - + - + - + - + TGF-pi with changes in the expression of a& or a& integrins (Fig. " " "--" 4), even though the differentiated cells retained TGF-/3 recep200 tors (not shown). The present results extend previous work on theregulation of integrins by showing that the expression of vitronectin Ireceptors and LFA-1is susceptible to control by TGF-B1. The results show that vitronectin receptor expression is sensitive to TGF-B1 action in fibroblasts andosteogenic sarcoma cells. 60 Exposure of these cells to TGF-/31 results in increased production of a& vitronectinreceptor complex. This action 45 subunit involves an increase in the steady-state level of I 1 I mRNA and possibly B3 subunit mRNA. The three cell lines 0

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FIG.4. Response of THP-1 monocyte and macrophage integrins toTGF-B1. Undifferentiated THP-1 monocytes, or macrophages obtained by treatment of THP-1 cells with 16 nM phorbol myristate acetate for 48 h, were incubated withmedium alone or with 250 PM TGF-8 for either 12 to 24 h in serum-free RPMI 1640 medium. The cultures were labeled with 20 pCi/ml ['%]cysteine in cysteinefree minimum essential medium during the final 3 h of incubation. The cells were harvested and detergent-extracted. The soluble cell extracts were treated with either an anti-@, subunit antisera or an anti-& subunit antisera. Immunecomplexes were recovered by binding toprotein A-Sepharose andanalyzed by electrophoresisand fluorography. Anti-S1 immunocomplexes were not reduced while the anti-& immunocomplexes were reduced with dithiothreitol prior to electrophoresis. Solid arrows denote the B subunits (and 81 subunit biosynthetic precursor);open arrows indicate therespective coprecipitating a subunits (asand QL).

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target for TGF-/31 action (17-19). THP-1 is a human monocytic leukemia cell line that grows in suspension butacquires FIG.5. TGF-8 elevates expressionof a~in THP-1 monocyte. a substratum-adherent macrophage phenotype after induction THP-1 monocytes were incubated with or without 250 PM TGF-Dl with the phorbol ester, phorbol myristate acetate(26). for 9 h in serum-free RPMImedium and then incubated incysteineUndifferentiated THP-1 cells were responsive to TGF-/31 free minimum essential medium containing 20 pCi/ml ["S]cysteine as assessed by the effect of this factor on the level of products with or without TGF-81 for an additional 3 h. The cells were harprecipitated with anti-P1 subunit antibody (Fig. 4). Based on vested, extracted with detergent,andimmunoprecipitated witha mouse monoclonal antibody specific for the (YL subunit. The immune the resultsof pulse-chase metabolic labelingexperiments (not complexes were recovered by incubation with goat anti-mouse IgG shown), these products were identified as the100-kDa biosyn- and protein A-Sepharose. The precipitates were analyzed by electrothetic precursor of the p1 integrin subunit, the 130-kDa ma- phoresis on polyacrylamide gels under reducing conditions and fluoturesubunit,and a 150-kDa a subunit(s)that coprecipi- rography.

TGF-0 Regulates 0 2 and 0 3 Integrins

392

examined here, WI-38, MG-63, and 3T3-L1, also respond to TGF-P1 with marked changes in the expression of a& integrins, and these changes follow kinetics similar to those of the vitronectin receptor response (17,19). Theseobservations suggest the presence of common regulatory mechanisms in the expression of aPI and aP3integrins. These two families of integrins are functionally more closely related to each other thanto integrins in that they function as extracellular matrix receptors rather than as mediators of intercellular adhesion. The similarity between crPl and aP3 integrins may extend to other regulatory properties, as well. For example, their p subunits conserve in the same amino acid sequence position a cytoplasmic tyrosine residue that may function as a regulatory phosphorylation site (10, 27). Phosphorylation of & subunit on tyrosine does indeed occur in cells transformed by oncogenes that encode proteins with tyrosine kinase activity (28). Despite these similarities, the ligand binding specificity of the vitronectin receptor and its differential location relative to other integrinsin adhesion plaques of cells in culture (29) suggest that the vitronectin receptor plays a distinct role in cell adhesion that can be modulated by TGFP action. The response of THP-1 leukemia cells to TGF-P1 is characterized by an elevation of LFA-1 a subunit synthesis without a detectable change in the level of the Pz subunit. This effect occurs concomitantly with the up-regulation of an ab1 integrin, probably the fibronectin receptor in these cells, and is lost upon THP-1 cell differentiation into the macrophage phenotype. The resistance of PZ subunit expression to regulation by TGF-pl has been observed in two other lymphoid cell lines (U937 and HL-60) and contrastswith the responses of PI and P 3 integrin subunit expression to TGF-P action. These results suggest that TGF-P1 action on certain types of leukocytes may regulate both the adhesion of these cells to stromal substrata, and the interaction with other cells of the immune system via LFA-1 cell adhesion receptors. These effects of TGF-e1 might be relevant in the role of TGF-P as a powerful immunosuppressive lymphokine (20,21). Acknowledgments-We gratefully acknowledge Drs. R. McEver, T. Springer, and E. Ruoslahti for providing antibodies and cDNAs for these studies. We thank John Rauth for dedicated help with cell culture and Catherine English for preparation of the manuscript.

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