Comparative Haematology International (1998) 8:142-149 © 1998 Springer-Vertag London Limited
COMPARATIVE HAEMATOLOGY INTERNATIONAL
Original Article Tenascin-C is Synthesised and Secreted by Megakaryocytes, whose Adherence to Intact Tenascin is Mediated by the Integrin Subunit fil F. Tablin ~, M. J. Rabier ~, N. J. W a l k e r ~, V. M. Velasco l, C. L. Field j and R. M. L e v e n 2 iDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA; 2Department of Anatomy, Rush Medical College, Chicago, IL, USA
Abstract. Bone marrow megakaryocytes reside on a complex extracellular matrix, produced by stromal cells, as well as by megakaryocytes themselves. Using immunofluorescence microscopy we have determined the distribution of tenascin-C in guinea-pig bone marrow, finding that it has a prominent fibrillar distribution in association with megakaryocytes. Western blot analysis of CHRF-288 cells, a human megakaryocytic cell line and isolated purified guineapig megakaryocytes, with polyclonal and monoclonal antibodies, demonstrate the presence of three tenascin isoforms (210, 220 and 250 kDa). Immunoprecipitation of radiolabelled isolated guinea-pig megakaryocytes cultured on a type I collagen gel, demonstrate that these three tenascin isoforms were present in megakaryocytes, whereas only two isoforms were secreted into the media (210 and 220 kDa). Analysis of rat bone marrow by in situ hybridisation reveals that transcripts for tenascin-C are present in megakaryocytes, Integrin regulation of CHRF cell adhesion to intact tenascin was shown to be mediated solely by the fl~ integrin. Keywords: CHRF cells; Extracellular matrix; Integrins; Megakaryocytes; Tenascin-C
Introduction In vivo, megakaryocytes lie adjacent to the bone marrow sinus wall, and reside on a complex extracellular matrix Correspondence and offprint requests to: Dr Fern Tablin, Department of Anatomy, Physiology & Cell Biology, School of Veterinary Medicine. 1321 Haring Hall, University of California - Davis, Davis, CA 95616, USA.
composed of fibronectin, laminin, and type IV collagen (Bentley et al. 1981; Topp and Tablin 1991: Clark and Keating 1995). All of these matrix molecules as well as matrix molecules produced by bone marrow stromal cells (Zuckerman and Rhodes 1985; Klein 1995) form an adhesive microenvironment for the megakaryocyte. Tenascin is a large ( > 1900 kDa) extracellular matrix oligomeric glycoprotein, that is postulated to play a role in morphogenetic movement of ceils and subsequent tissue development and organisation. At its amino terminus (in mouse and human), there is a cysteine-rich segment, followed by 14.5 epidermal growth factor (EGF)-like repeats, 16 fibronectin type III repeats and a globular carboxyl terminus which is homologous to fibrinogen (Erickson and Bourdon 1989; ChiquetEhrismann 1995). The cDNA sequence of tenascin from chicken, mouse and human, has shown that as a result of alternative splicing, there are three main tenascin isoforms, the synthesis of which appears to be under specific developmental control (Chiquet-Ehrismann et al. 1991; Sage and Bornstein 1991 ; Sriramarao et al. 1993). Antibodies to tenascin recognise these isoforms with molecular masses of 250, 220 and 210 kDa in immunoblots of mammalian tissues. Tenascin is present in two types of tissues, The smallest form of tenascin is present in the adult connective tissue of myotendinuous junctions and the gizzard; both areas requiring high tensile strength (Chiquet and Fambrough 1984; ChiquetEhrismann et al. 1991). Tenascin is also highly expressed in developing tissue, where cell migration and extracellular matrix reorganisation occur. Tenascin is present in the early embryo (Mackie et al. 1987; Prieto et al. 1990) and has been observed to decrease cell adhesion and promote cell migration (Friedlander et al. 1988; Halfter et al. 1989; Lotz et al, 1989; Prieto et al. 1990;
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Tenascin-C in Megakaryocytes Riou et al. 1990) suggesting that tenascin can function as both a positive and negative modulator of cellsubstratum adhesion (Chiquet-Ehrismann et al. 1988; Gotz et al. 1996). Tenascin also has been shown to be an important regulator of cell motility during neurite outgrowth (Wehrle and Chiquet 1990). In adult tissues, other than those associated with the musculoskeletal system; tenascin expression is more limited, being restricted primarily to regions of continuous renewal, such as the crypts of the intestine (Weller et al. 1991). Tenascin is produced by cultured human bone marrow stromal cells (Klein et al. 1993) and increased tenascin immunoreactivity is observed in human bone marrow in association with marrow fibrosis and conditions resulting in megakaryocytic hyperplasia (Soini et al. 1993). As part of our ongoing effort to understand the interaction between megakaryocytes and their matrix environment and to determine the role of matrix molecules in proplatelet formation, we have investigated the synthesis, secretion and distribution of tenascin in bone marrow megakaryocytes and CHRF-288 cells.
Materials and Methods Immunofluorescence Bone Marrow. Bone marrow was obtained from long bones of either male Hartley guinea pigs or SpragueDawley rats and fixed in 1% paraformaldehyde in phosphate buffered saline (PBS) pH 7.2 for 1 h at room temperature. Marrow was embedded in OCT embedding compound (Miles, Elkhart, IN) and snap frozen in liquid nitrogen. Samples were stored at -80°C. Sections (10 #m) were cut, washed in PBS, and blocked in 10% donkey serum in PBS overnight. Sections were then incubated with anti-tenascin polyclonal rabbit antibody (GIBCO-BRL, Grand Island, NY) and a secondary donkey anti-rabbit IgG conjugated to TRITC (Amersham Corp., Arlington Heights, IL). Control slides were incubated with non-immune rabbit IgG and secondary antibody.
CHRF-288 Cells. CHRF-288 cells were the gift of Dr Michael Lieberman, University of Cincinnati and were maintained in minimal essential medium with Earle's salts and 10% fetal bovine serum (GIBCO-BRL). These cells are a human megakaryoblastic leukaemic cell line which can be stimulated by phorbol myristate acetate (PMA) to undergo differentiation, to become polyploid, and to express megakaryocytic gene products (Dorn et al. 1994). The experimental studies, CHRF cells were cultured in serum-free media and stimulated with 10 ng/ ml PMA. Cells were grown on Lab-tech slides (Nunc Inc., Naperville, IL) in the presence and absence of PMA for 48 h, fixed in 1% paraformaldehyde, permeabilised in methanol and stained with an anti-human tenascin monoclonal antibody (Calbiochem, LaJolla, CA) and a secondary goat anti-human IgG TRITC (Amersham
Corp., Arlington Heights, IL). Control cells were stained with non-immune IgG and secondary antibody.
Immunoprecipitation and [~sS]Methionine Labelling Guinea-pig megakaryocytes were isolated by immunomagnetic bead separation as previously described by Leven and Rodriguez (1991). Megakaryocytes were cultured on a type I collagen gel, and incubated with 200 #Ci/ml of [35S]methionine (SJ 1015, Amersham Corp., Arlington Heights, IL) for 18 h. At 18 h, the medium was removed and any non-adherent cells were removed by centrifugation. Adherent cells were washed with cold medium and were extracted by the addition of a lysis buffer (150 mM NaC1, 50 mM Tris HC1, lmM EGTA, 1 mM phenylmethyl sulphonyl fluoride, 1 pg/ml aprotinin, 1 #g/ml leupeptin with 1% Triton X-100). Solubilised cellular material was added to the cell pellet recovered from the medium and the total cell lysate was centrifuged at 13000 g to remove insoluble material. From this point on cell lysates and media were processed identically. Material was pre-cleared by incubation with normal IgG to remove protein which are non-specifically 'sticky' followed by precipitation with protein-G agarose. This complex was removed by centrifugation and the lysate was incubated with anti-tenascin polyclonal antibody (GIBCO-BRL, Grand Island, NY) followed by precipitation with protein-G agarose. The agarose beads were removed by centrifugation, washed thrice in lysis buffer and suspended in Laemmli sample buffer (Laemmli 1970). Proteins were separated on a 7.5% reducing polyacrylamide gel and labelled proteins in the immunoprecipitate was visualised by autoradiography with Kodak X-Omat film.
Western Blot Analysis Megakaryocyte and CHRF cell (with and without PMA stimulation) lysates were prepared by boiling cells in equal volumes of 2 x Laemmli sample buffer (Laemmli 1970). Serum-free culture medium from CHRF cells was concentrated in Centricon 30 (Amicon, Beverly, MA) and mixed with an equal volume of 2 x Laemmli buffer. Samples were run on a 7.5% sodium dodecyl sulphate (SDS)-polyacrylamide gel. Proteins were transferred to nitrocellulose (Towbin et al. 1979), blocked in 10% nonfat dry milk in Tris-buffered saline and probed with either a monoclonal (Calbiochem, LaJolla, CA) or a polyclonal (GIBCO-BRL, Grand Island, NY) antibody to tenascin. The blots were washed and reacted with a secondary antibody conjugated to horseradish peroxidase. The chromogenic substrate was 4-chloro-l-naphthol.
bz Situ Hybridisation In situ hybridisation was performed according to a modification of the procedure of Xu et al. (1995) with
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the exception that the c D N A probes were conjugated to digoxygenin (Boehringer Mannheim Corp., Indianapolis, IN) according to the manufacturer's directions. The digoxygenin was recognised with anti-digoxygenin antibodies according to the method of Karr et al. (1995). Tenascin c D N A was the gift of Dr Richard Tucker (Tucker et al. 1993). fi-actin c D N A served as a positive control, and bacterial c D N A (pBR 322) was used as the negative control.
Cell Adhesion Assays The adhesion assay was performed according to the methods of Chemicon International Inc. (Temecula, CA). Briefly, C H R F cells were grown in serum-free media and stimulated with PMA 24 h prior to the assay. Precoated 96-well plates coated with tenascin (CytoMatrix Cell Adhesion Strips; Chemicon International Inc.) were washed in media and blocked with 1% BSA for 30 rain at 37'C. A single C H R F cell suspension was prepared without the use of an enzymatic digestion buffer and cells were incubated either with an antibody ( c o n t r o l ) , n o n - i l n m u n e lllOklSe a l l d / o r rat l g a , or monoclonal antibodies to fiL (AIIB2 (Hall et al. 199[)) the gift of Dr Caroline Damsky), ct~[]3 (LM 609 (Cheresh 1987) Ihe gift o[" Dr David Chercsh of ~,[¢5 (PI56
(Wayne et al. 1991) Chemicon, Temecula, CA) at a concentration of 10 itg/ml, for 2 h at 37°C. All three of the monoclonal antibodies used are well characterised, and each has been shown to be a function-blocking antibody. AIIB2 (the monoclonal antibody to the /31 integrin) inhibits adhesion to fibronectin, laminin, and collagen (Hall et al. 1990). A suspension (100/d) of 106 cells/ml was plated in each well, and incubated at 37°C for 45 rain. Plates were washed three times in PBS and incubated in 100 ill of 0.2% crystal violet in 10% ethanol for 5 min at room temperature. Plates were washed three times in PBS, and incubated with 100 #l of 1% SDS in PBS for 5 rain at room temperature and were read on a microtitre plate reader (Model E1 340 Biokinetics plate reader, Bio-Tek Instruments, Winooski, VT at 540 nm). Samples were run in triplicate.
Results
Tenascin hnmunoreactivitv is Present in CHRF Cells and Guinea-pig Bone Marrow Frozen scclions of guinea-pig bone marrow slained with a polychmal antibody to lenascin demonslrate that few positively slained fibrils arc randomly distributed throughout the haematopoietic spaces. However, a prominent lihrillar paUern is present in association with
1
A~
Fig. 1. Tenascin-C in guinea-pig bone marrow. A Phase, B fluorescence: scant tenascin staining throughout, with fibrils specifically associated with
megakaryocytes. Most megakaryocytes stained positively by the antibody (arrows).C Phase, D fluorescence: bone marrow arteriole (positive control).
Tenascin-C in Megakaryocytes
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CHRF-288 cells grown in serum-free media with and without the use of PMA to upregulate ploidy and megakaryocyte-specific gene products also were examined for tenascin immunoreactivity. Cells grown in the absence of PMA are not stained by a monoclonal antitenascin antibody (data not shown), however, PMA stimulated cells are strongly stained by the antibody (Fig. 2). Staining is diffusely present within the cytoplasm of these cells, as well as in a distinctive perinuclear distribution (Fig. 2B), with many cells exhibiting a fine positively staining punctate fluorescence. Extracellular fibrillar material secreted by the CHRF cells is also stained by the monoclonal antibody (Fig. 2A).
Megakaryocytes Contain Multiple Isoforms of Tenascin-C
Fig. 2. PMA stimulated CttRF ceils. A Fine fibrillar extracellular matrix labelled by the anti-tenascin antibody. B Diffuse cytoplasmic and bright perinuclear staining, with some fine punctate fluorescence. C Phase contrast microscopy of the fibrillar meshwork.
megakaryocytes (Fig. 1 A, B) which also are stained by the antibody. Tenascin staining is also present in association with arterioles (where its synthesis by smooth muscle cells has previously been reported (Mackie et al. 1992) (Fig. 1 C, D). Control sections of guinea-pig man-ow incubated with a non-immune IgG showed no staining (data not shown).
Western blot analysis of PMA stimulated and unstimulated CHRF cell lysates and their media, probed with a monoclonal anti-tenascin antibody, demonstrates that there is a differential distribution of the protein. PMA stimulated ceils (Fig. 3A, lane 2) and their media (Fig. 3A, lane 3) have three immunoreactive bands, with molecular weights of 250, 220 and 210 kDa. Isoforms having the same molecular mass have previously been identified in other systems (Chiquet-Ehrismann et al. 1991; Chiquet and Fambrough 1984). Unstimulated CHRF cells (Fig. 3A, lane 4) and their media (Fig. 3A, lane 5) do not contain tenascin detectable by Western blot analysis. Guinea-pig megakaryocyte cell lysates analysed by SDS-PAGE and Western blotting using a polyclonal anti-tenascin antibody (Fig. 3B) demonstrated the presence of three high molecular weight bands (210, 220 and 250 kDa). Additional pale-staining lower molecular weight bands are present, which likely present proteolytic degradation of the higher molecular weight form of tenascin previously described by Siri et al. (1995). Isolated guinea-pig megakaryocytes were cultured on a type I collagen gel in the presence of [35S]methionine. The soluble media and the megakaryocytes were immunoprecipitated with a polyclonal anti-tenascin antibody (Fig. 3C). Two isoforms with molecular masses of 210 and 220 kDa were detected in the media (Fig. 3C, lane 1'), while three isoforms (210, 220, 250 kDa) were present in megakaryocyte lysates (Fig. 3C, lane 2'). In addition, multiple bands, suggestive of proteolytic degradation are present within the samples.
Bone Marrow MegakaJ~ocytes Express mRNA for Tenascin Rat bone marrow was fixed and processed for in situ hybridisation using a cDNA to rat tenascin (the gift of Dr Richard Tucker (Tucker et al. 1993), conjugated to digoxygenin. A moderate level of tenascin message was
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present in megakaryocytes, and was characterised by punctate staining within the cytoplasm (closed arrows) (Fig. 4A). Abundant hybridisation signal was present in endothelial cells of the vascular sinuses (open arrows) (Fig. 4A). //-actin positive controls demonstrated an abundance of message in endothelial cells, megakaryocytes and other haematopoielic cells (Fig. 4B).
Megakarvocyte Adhesion to Tenascin is Mediated by [I/ lntegrin To evaluate the role of integrins in mediating the adhesion of megakaryocytes to a tenascin matrix, CHRF cells, with and without PMA stimulalion, were allowed to adhere to tenascin coated plates (Fig. 5). Unstimulated cells poorly adhered to tenascin, whereas PMA-treated cells demonstrate strong adherence. Adhesion is not blocked by function blocking monoclonal antibodies to the vitronectin receptors C~v[]3 (Cheresh 1987) or ~v/35 (Wayner et al. 1991), but is completely abolished when the PMA-treated cells were preincubated with a wellcharacterised function blocking monoclonal antibody to [~1 (Hall et al. 1990). Unlike some other cell adhesion systems previously examined (Sriramarao et al. 1993), no synergism was seen between the vitronectin receptor integrins and the [4~ integrin.
Discussion The results presented here demonstrate that tenascin-C is expressed by both CHRF-288 cells and guinea-pig megakaryocytes in vitro. [35S]Methionine labelling, immunoprecipitation, and ilnmunocytochemistry show that tenascin is present within megakaryocytes and it secreted into the soluble media as well as being secreted as a fibrillar matrix. Three tenascin isoforms (210, 220 and 250 kDa) were identified by Western blot and immunoprecipitation. These isoforms, of the same molecular mass previously reported by other researchers (Chiquet-Ehrismann et al. 1991; Sage and Bornstein 1991; Sriramarao et al. 1993) are most likely the result of alternative splicing, although their role in development
m
I Fig. 3. hnmunoblot and [35S]methionine analysis. A CHRF cells and serum-free media, with and without PMA Lane 1, molecular weight markers; lane 2, PMA-treated CHRF cells; lane 3, PMA media; lane 4, unstimulated CHRF cells; lane 5, unstimulated CHRF cell media. B Megakaryocyte cell lysates. C [35S]Methionine labelled guinea-pig megakaryocytes. Lane 1', media; lane 2', megakaryocytes.
in general and in megakaryocyte development in specific remains unclear. Tenascin message and protein are also present in megakaryocytes in vivo. Megakaryocyte adhesion to intact tenascin is modulated solely by the integrin [/1. Tenascin-C is strongly expressed in embryonic and developing tissues, where it has been shown to be both an adhesive and an anti-adhesive protein (ChiquetEhrismann et al. 1991; Spring et al. 1989; Prieto et al. 1992). In vitro tenascin binds to immobilised fibronectin (Friedlander et al. 1988" Chiquet-Ehrismann et al. 1988) and to cell surface heparan sulphate proteoglycans (Prieto et al. 1990). These cell surface proteoglycans also recognise fibronectm (Salmivitra et al. 1991; Damsky and Werb 1992). The function of tenascin in the reorganisation of tissues is unclear; however, tenascin has been shown to collaborate with fibronectin in upregulating collagenase gene expression in synovial fibroblasts (Tremble et al. 1994). This association has been suggested to function in the regulation of gene expression in regions of tissue undergoing phenotypic changes. During normal megakaryocyte maturation, megakaryocytes reside on the abluminal aspect of the vascular sinus endothelium (Mohandas and Prenant 1978), where they extend long beaded processes into the vascular sinus (Weiss and Chen 1975; Lichtman et al. 1978), a process referred to as proplatelet formation. Bone marrow stromal cells produce the localised abluminal haematopoietic microenvironment which consists oK but is not limited to: tenascin, fibronectin (Weiss and Reddy 1981; Reilly et al. 1985) and perlecan (a heparan sulphate proteoglycan) (Klein et al. 1994). Recent studies have demonstrated that guinea-pig megakaryocytes also express an embryonic form of fibronectin (Schick et al. 1996). Fibronectin and tenascin - large multidomain molecules - are also recognised by cellular integrin receptors. Recently, the 0~2fl] and avfl3 integrins have been demonstrated to mediate the adhesion of endothelial cells to tenascin (Joshi et al. 1993; Sriramarao et al. 1993). The vitronectin receptor a,,fl3 recognises the RGD site in the third type III repeat of tenascin (Bourdon and Ruoslahti 1989), and ~2/~1 may recognise the carboxyl
Tenascin-C in Megakaryocytes
147 Adhesion of CHRF Cells to Tenascin 0.5
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Fig. 5. Adhesion of CHRF cells to tenascin. The adhesion units are arbitary numbers, based on optical density.
Fig. 4. In situ hybridisation with cDNA to rat tenascin. A Strong hybridisation signal in endothelial cells of the vascular sinuses (open arrow), moderate punctuate signal in the cytoplasm of a megakaryocyte (closed arrow). B Abundant hybridisationsignal in virtually all haemopoietic cells with the positive control (fi-actin), with a signal strong enough to produce a dark blue ring in the cytoplasm of a megakaryocyte (arrow). C No hybridisation signal in the negative control (pBR 322). terminal fibrinogen-like domain (Joshi et al. 1993). Megakaryocytes express numerous integrins including, but not limited to: ~2fil, ~Sfil, ~6fil, ~d33, and GP IIb-IIIa (Hemler et al. 1988; Lam et al. 1989; Topp and Tablin 1991). We have shown that megakaryocyte adhesion to intact tenascin is mediated solely by the integrin ill.
The process of proplatelet formation can be simulated in vitro by culture of either whole bone marrow (Radley and Scurfield 1980) or of isolated, purified megakaryocytes grown on a variety of matrix proteins (Handagama et al. 1987; Tablin et al. 1990; Topp et al. 1990; Leven and Tablin 1992; Choi et al. 1995a,b). Megakaryocytes grown in this manner frequently undergo profound morphological change. Initial cellular adhesion to matrix is accompanied by the development of focal contacts, (Tablin et al. 1990: T o p p e t al. 1990), as well as the assembly of actin filaments at focal contact sites and reorganisation of the actin rich peripheral zone (Zucker-Franklin and Petursson 1984; Stenberg and Levin 1987, 1989; T o p p e t al. 1990). Subsequent to these initial events, megakaryocytes extend long beaded processes, which are no longer adherent to the matrix, but which can be observed to float 'free" in the culture media, as proplatelet processes, which subsequently fragment to form functional platelets (Choi et al. 1995a,b). Proplatelet formation in vitro requires extensive cytoplasmic and plasma membrane reorganisation, accompanied by loss of the megakaryocyte's association with the matrix. Alterations in matrix associations may be due to a variety of factors, the most likely of which is the production of matrix metalloproteinases. Several studies have demonstrated that tenascin is susceptible to degradation by these metalloproteinases (Klein et al. 1993: Imai et al. 1994). Cultured megakaryocytes secrete several matrix metalloproteinases (Leven and Yee 1990). Production and secretion of these megakaryocyte metalloproteinases could be upregulated in a manner similar to that of synovial fibroblasts. In vitro tenascin and fibronectin collaborate to upregulate synovial fibroblast synthesis and secretion of matrix metalloproteinases (Tremble et al. 1994). Tenascin and fibronectin may act in an analogous fashion to upregulate the production and secretion of megakaryocyte matrix
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metalloproteinases. These metalloproteinases could then presumably disrupt integrin-matrix interactions, allowing for the megakaryocyte's cytoplasmic processes to elongate in the absence of adhesive forces, resulting in the beading of proplatelets and their eventual fragmentation and platelet formation. Acknowledgements. We thank Dr Richard Tucker for the gift of rat tenascin eDNA. We also thank Dr Carolyn Damsky for the gift of the AIIB2 antibody. This work was supported by grants from the American Cancer Society (No. DHP-119, to RML), the Naval Research and Development Command (N000014-94-1-0379) IF.T0 and the Genetech Foundation (VV).
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