Internalization and Intracellular Processing of Bone Morphogenetic Protein (BMP) in Rat Skeletal Muscle Myoblasts (L6)

Cell. Signal. Vol. 9, No. 1, pp. 47–51, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0898-6568/97 $17.00 PII S0898-6568(96)00094-0

Internalization and Intracellular Processing of Bone Morphogenetic Protein (BMP) in Rat Skeletal Muscle Myoblasts (L6) Leena Jortikka,*†‡ Minna Laitinen,†‡ T. Sam Lindholm†‡§ and Aulis Marttinen§ †Bone Transplantation Research Group, Institute of Medical Technology, University of Tampere, 33101 Tampere, Finland, ‡Department of Orthopaedics and Traumatology, University Hospital of Tampere, 33520 Tampere, Finland, and §Medical School, University of Tampere, 33101 Tampere, Finland

ABSTRACT. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b (TGF-b) superfamily capable of inducing bone and cartilage formation in ectopic extraskeletal sites and transducing their effects through binding to serine-threonine kinase receptors. In this study, the fate of 125I-labelled native BMP after binding to cell surface receptors on L6-myoblasts was examined with both continuous and intermittent exposure of the ligand. BMP was readily internalized in L6 cells at 1378C, and the internalization reached a plateau in 2 h. Intracellular degradation of 125I-labelled BMP was established, and degradation products were also detected in binding buffer, indicating exocytosis of the processed ligands. BMP receptors were shown to be subject to acute down-regulation by the ligand, and receptors were completely recycled in 3 h. Hence, we conclude that BMP receptors, like receptors for various other polypeptide ligands, have the ability to mediate intracellular delivery and degradation of the ligand. Copyright  1997 Elsevier Science Inc. cell signal 9;1:47–51, 1997. KEY WORDS. BMP, Bone morphogenetic protein, Internalization, Degradation, Receptor recyclation

INTRODUCTION Bone morphogenetic proteins (BMPs) are members of the transforming growth factor b (TGF-b) superfamily, playing important roles in the regulation of cell growth and differentiation. During the last decade bone morphogenetic activity has been purified from bone matrix of various animals, and in addition to these native BMPs, at least nine BMPs with TGF-b gene-related sequences have been reported [1–3]. Although BMP activity was first defined by implanting demineralized bone matrix at ectopic sites in vivo, the effects of these proteins on various cell lines have since been established, indicating initiation, promotion and maintenance of the osteogenic phenotype in vitro [4–8]. Recent studies have also demonstrated that besides taking part in chondrogenesis and osteogenesis, BMPs are also involved in embryonic skeleton formation [9–11]. After characterizing the proteins themselves, research has focused on identifying specific cellular binding proteins for BMPs and determining the molecular events by which BMPs induce formation of bone and cartilage as well as their other actions [12, 13]. The first isolated BMP receptor, daf-4, was shown to bind both BMP-2 and BMP-4, when expressesd in monkey COS cells [14]. This finding con*Address correspondence to Leena Jortikka, M.D., Institute of Medical Technology, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland. E-mail: [email protected] Received 7 January 1996; and accepted 1 March 1996.

firmed that BMPs, like other members of the TGF-b superfamily (i.e., TGF-b and activins), exert their effects through single-pass transmembrane proteins with a serine/ threonine kinase domain on the cytosolic side of the plasma membrane. At least two types of transmembrane serine/ threonine kinases, receptors I and II, induce biological signals of the TGF-b superfamily members, type II receptors acting as primary receptors and type I receptors as their substrates propagating the signal. However, there is conflicting evidence concerning signal transduction by BMPs, since various BMP receptors have been shown to be able to bind the ligand on their own, indicating a simpler receptor scheme than those of TGF-bs or activins, which require formation of heteromeric kinase receptor complexes between receptor type I and II for signal transduction [15–18]. During the past 20 years, research in the field of growth factors and regulators has been extensive. Their biological effects are induced by binding to cell surface receptors, and in many cases, the structure and nature of membrane binding sites have been elucidated [19]. It is generally agreed that after binding to the receptor on the target cell surface, cellular signaling of biologically active factors either occurs via a so-called second-messenger through the cell membrane, or the factor can also be internalized into the cell by the receptor. In the case of BMPs, the mechanisms involved in signal transduction have not been extensively explored. In the pathway of receptor-mediated endocytosis, the ligand is internalized via coated pits and transported to the mem-

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brane system of the endosomal compartment. Most ligands dissociate from their receptors in the endosome and are finally degraded in lysosomes, while the receptors can be recycled back to the cell surface for reuse or degraded with their ligands in lysosomes [20]. The present study was designed to explore whether native BMP is endocytosed and intracellularly degraded after receptor binding in rat skeletal muscle myoblasts (L6), which, to conclude from our previous studies, are target cells for the action of BMP [21].

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mented with 10% foetal bovine serum (FBS, Gibco, Paisley, Scotland) and antibiotics (50 IU/mL penicillin and 50 mg/ mL streptomycin, Gibco, Paisley, Scotland) in a humid atmosphere of 5% CO2 and 95% air at 1378C. Cells were subcultivated before confluency and differentiation, and for the experiments the monolayers were cultured for 2–3 days, nearly to the confluent stage. Internalization of BMP

MATERIALS AND METHODS Bone Morphogenetic Protein Highly purified BMP was obtained from demineralized bovine bone matrix as previously described [22]. Briefly, noncollagenous protein material was extracted in 4M GuHCl and water- and citrate buffer-insoluble material collected, solubilized in 6 M urea and fractionated by preparative isoelectric focusing. The osteoinductive, Tris buffer-soluble material was then fractionated by HPLC gel filtration and lyophilized. Bone-inducing activity was confirmed by intramuscular implantation of the purified protein in mice. Iodination of BMP BMP was iodinated by the chloramin T method [23]. In brief, 125 mg of BMP in 250 mL of 6 M urea was diluted with 1.25 mL of 0.1 M Tris-HC1 buffer, pH 7.2. Then 200 mCi of 125I (Amersham, Buckinghamshire, UK) was added to the reaction mixture and the reaction started by adding one Iodo Bead (Pierce, Rockford, IL, USA) to the solution. After 5 min, the reaction was terminated and radiolabelled BMP separated by passing the reaction mixture through a PD-10 gel filtration column (Pharmacia, Uppsala, Sweden) pre-equilibrated with 0.1 M Tris buffer, pH 7.2. Before further experiments, noncovalently bound 125I was removed by re-passing the mixture through a PD-10 column with 0.07% CHAPS, 0.02% octylglycopyranocid, 10% 2-propanol and 2% HCOOH in 0.1 M Tris buffer, pH 7.2, as the eluent. As previously, the 125I-labelled BMP preparation was analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) on a 12% acrylamide gel under reducing conditions followed by autoradiography. The gel revealed a broad band below 20 kD after reduction corresponding to the ionidated monocomponent protein material. However, three minor bands with MW above 50 kD were also identified, suggesting that despite reducing conditions, the BMP complex did not completely dissociate to monocomponent form. Biological activity of BMP after radioiodination was confirmed on the basis of its dose-dependent induction of 45Ca incorporation and stimulation of ALP activity in L6 cells by methods previously described [21]. L6 Cell Culture L6 rat skeletal muscle myoblasts were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Paisley, Scotland) supple-

To study events after receptor binding, the cultured myoblasts (2 3 106 cells on each dish) were first incubated with 125I-BMP in 4 mL of Hanks’ balanced salt solution containing 20 mM HEPES and 0.2% bovine serum albumin (binding buffer) at 1108C for 2 h in order to occupy the receptors with the ligand. To start internalization, the incubation was continued at 1378C in binding buffer without BMP, or with 125I-labelled BMP plus a 5-fold concentration of unlabelled BMP to minimize dissociation of ligand from receptor. After various incubation periods, the cell cultures were washed twice with Hanks’ and harvested with 0.25% trypsin-EDTA (Gibco, Paisley, Scotland). The pellet was then treated with 0.5 M citrate buffer, pH 2.9, for 1 min, to remove uninternalized ligand from cell-surface receptors. After centrifuging, the supernatant was discarded and the radioactivity of the cells counted to determine whether the ligand had internalized in myoblasts. Degradation of BMP Next, based on an assumption that BMP is degraded during incubation at 1378C, we determined degradation products accumulated in binding buffer and associated in cells. When studying whether BMP is released to the binding buffer after degradation, samples of 400 mL of the binding buffer were assayed at various time points. Aggregates of degraded BMP were dispersed with 0.02% octylglycopyranocid, 5% HCOOH, 0.07% CHAPS, and 10% 2-propanol. Native 125I-BMP was precipitated with 10% trichloracetic acid (TCA, final concentration) and the radioactivity of the TCA-soluble material was counted. As a control the degradation of BMP was measured by incubating equal amounts of 125I-BMP in binding buffer without cells. To quantitate cell-associated BMP products, cells were lysed with 0.05 M sodium-phosphate buffer, containing 150 nM NaC1, 0.1% octylglycopyranocid, 1% Nonidet P-40, 0.1% CHAPS, and 15% 2-propanol, and treated with TCA as above. Receptor Down-Regulation and Recyclation The myoblasts were preincubated with a high concentration of unlabelled BMP at 1108C for 2 h to occupy all the receptors with the ligand. After washing twice with cold Hanks’, the incubation was proceeded in binding buffer at 1378C for different periods of time to start the internalization of the ligand. Finally, the cultures were cooled at

Intracellular Processing of BMP

FIGURE 1. Internalization of

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I-BMP in L6 myoblasts. After preoccupying the receptors with the radiolabelled ligand at 1108C for 2 h, the incubation was continued at 1378C either with 125I-BMP containing media (triangles) or with BMP-free media (squares). At each time point the cells were harvested, cell-surface-bound ligands removed from the receptors and the radioactivities of internalized ligands determined. Nonspecific binding was subtracted from the raw data. A duplicate assay was repeated with two different iodinated preparations and the results were reproducible.

1108C, 125I-BMP was added to the culture media and incubation was continued for 2 h. Binding of 125I-BMP on myoblast surface was determined as recently described [21]. As a reference, cells not pretreated with unlabelled BMP were incubated with 125I-BMP in parallel. RESULTS Internalization of BMP Figure 1 summarizes cell-associated radioactivities after removing cell surface-bound labelled ligand with citrate buffer. In preliminary experiments it was demonstrated that citrate buffer was capable of removing essentially all radioactivity from the cell surface (data not shown). A rapid internalization of receptor-bound BMP was observed at 1378C. With continuous exposure to high concentrations of BMP, no statistically significant increase was found after 2 h. During incubation of cells with receptor-bound BMP in BMP-free media, internalization diminished after 30 min in consequence of the lack of unprocessed ligands.

125 I-BMP in L6 myoblasts. The amount of degraded 125I-BMP inside the cells and in the binding media was estimated by TCA precipitation. The data represent the total amount of degraded BMP in cells incubated in 125I-BMP containing media (triangles) or with BMP-free media (squares). BMP degraded in medium without cells was subtracted from the raw data. A duplicate assay was repeated with two different iodinated preparations and the results were reproducible.

FIGURE 2. Degradation of

loaded with unlabelled BMP reached the level of that on cells without pretreatment in 3 h incubation at 1378C. DISCUSSION In the present study, both internalization and intracellular degradation of BMP in rat skeletal muscle myoblasts (L6) were established, and BMP receptors on the surface of myoblasts were shown to be subject to acute down-regulation by the ligand. Consistent with past research with other polypeptide growth factors and morphogens, i.e., EGF (epidermal growth factor), PDGF (platelet-derived growth factor), and TGF-b [19, 24], our results indicate that receptor-bound BMP also undergoes rapid internalization at 1378C. With continuous exposure to the ligand at 1378C, internalization reached a plateau in two to three hours, reflecting a steady-state with transport into the intracellular interior

Degradation of BMP A final TCA concentration of 10% was found appropriate for precipitation of native 125I-BMP from degraded protein (data not shown). The radioactivities of the degradation products of 125I-BMP were determined from both intracellular interior of myoblasts and incubation media. As can be seen in Figure 2, the amount of radioactivity soluble in 10% TCA corresponding to degraded BMP increased during the observation time if myoblasts were continuously exposed to BMP at 1378C. However, with the lack of ligands, degradation also ceased after a rapid increase within the first 15 min. Down-Regulation and Recyclation of BMP Receptors The results in Figure 3 show that receptors first occupied with unlabelled BMP were for more than 1 h unable to bind 125 I-BMP. Nevertheless, binding of 125I-BMP on cells pre-

FIGURE 3. Down-regulation and recycling of the BMP receptor.

Near-confluent myoblast cultures were preincubated with unlabelled BMP at 1108C for 2 h to occupy the receptors with ligand; then after extensive rinsing, warm BMP-free media was added to the cells and the cultures were maintained at 1378C for various periods of time to allow endocytosis of the ligand. At each time point, the cultures were cooled to 1108C and 125 I-labelled BMP was added, and after 2 h incubation and harvesting, the cell-surface-bound radioactivities were counted. The broken line represents binding of 125I-BMP at 1108C without preincubation with unlabelled BMP.

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and release of degradation products back to the medium. In BMP-free media, internalization of BMP via receptors preoccupied with radiolabelled ligand occurred within 30 min, indicating fast endocytosis of the ligand; moreover, signal transduction of BMP appears to go through intracellular processing of receptor-bound BMP. Whether BMP is endocytoted alone or as a ligand-receptor complex as are most of the similarly acting factors, for example TGF-b [24], remains to be determined. However, even now the concept of endocytosis of the BMP-receptor complex must be taken into consideration in the case of developing new delivery systems for BMP in orthopaedic surgery or experimental orthopeadics. During the last decade BMP has been combined with numerous carriers (i.e., hydroxyapatite, tri-calcium phosphate ceramics, and different types of collagens) for the purpose of treating nonhealing fractures or congenital disorders [25]. On the basis of our findings it is clear that the bonds between the osteoinductive protein and the potential carrier material should not be tight and in no case covalent in order to allow endocytosis of the ligand and obtain the maximum benefit of the protein. Results of our BMP degradation experiments suggested that both the degradation and exocytosis of the processed ligands were very fast. Such a concept was supported by the minor amount of degradation products inside the cells (data not shown). Experiments carried through in BMP-free media with cells preincubated with radiolabelled ligand revealed that, in fact, the degradation and also almost simultaneous exocytosis of the degradation products occurred within the first 10–15 min after incubation at 1378C. It is not known whether the “purpose” of degradation is to enable exocytosis of already used BMP molecule or whether a certain fragment of degraded BMP acts as a second messenger and transfers the signal to the nucleus. By reason of the rapid exocytosis of degradation products the rate of degradation could be used to measure the activity of BMP receptors, and likewise, the sensitivity of cell or tissue to BMP in experimental conditions. The concept of down-regulation of growth factor receptors is widely accepted, meaning that continuous exposure to high concentrations of ligands decreases the number of cell surface receptors and the sensitivity of the target cell to the ligand [19]. However, in the case of TGF-b, there is conflicting evidence concerning receptor down-regulation. In BALB/c 3T3 fibroblasts, no significant change in the level of TGF-b receptors during exposure to TGF-b was detected, suggesting that these receptors, unlike most of the growth factor receptors, are not subject to acute down-regulation of the ligand [24]. Nevertheless, in kidney fibroblasts the receptor could be down-regulated by TGF-b to approximately 50% of the level initially observed [23]. Our results strongly support the conception that BMP receptors are down-regulated by BMP. We found that an increase in BMP binding on cells after exposure to a high concentration of BMP did not occur for almost one hour. Nevertheless, approximately complete receptor recyclation occurred during 3 h, apparently via recycling of already used, possibly internalized, BMP receptors or by an intracellular receptor

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pool or new receptor synthesis. These results suggest differences between TGF-b and BMP, members of the same superfamily, although some other recent findings, i.e., that only BMP is capable of inducing new bone formation in extraskeletal sites, have suggested that BMPs may have activities distinct from other TGF-b family members; hence also disparities in cellular kinetics would only be expected. Up to the present, the research in the field of BMP binding and signal transduction has concentrated on investigating the characteristics of the receptors found to be members of the serine/threonine kinase receptor family [13]. The results presented in this article demonstrate that the biological action of BMP also includes the pathway of intracellular ligand processing. In future studies it will be necessary to establish the physiological significance of BMP binding. Most likely one part of the binding occurs without any action, while another leads to intracellular signal transduction and nuclear response following differentiation into the osteoblastic lineage. A number of recent studies, as well as the present one, have shown that the response of BMP is a multistep, so far insufficiently characterized, process. Against this background, disturbances at any level can provoke modifications in target cell sensitivity to BMP, eventually leading to modified binding properties and, finally, to bone disorders. We must also give consideration to the potential role of BMPs in various other stages of organogenesis, in addition to osteogenesis and chondrogenesis, in improving our understanding of BMP action at the physiological level. Taken together, the present data indicate that BMP receptors have, in common with receptors for various other polypeptide growth factors and regulators, the ability after binding of ligand also to mediate intracellular delivery and degradation of BMP. Future research should be directed toward the intracellular events following internalization of the ligand and leading to nuclear response and differentiation of the target cell in the direction of bone cell. This study was financially supported by the University of Tampere and the Medical Research Fund of Tampere University Hospital.

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