Elsevier

Cellular Signalling

Volume 9, Issue 1, January 1997, Pages 47-51
Cellular Signalling

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

https://doi.org/10.1016/S0898-6568(96)00094-0Get rights and content

Abstract

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) 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 +37°C, 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.

Introduction

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) 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-β gene-related sequences have been reported 1, 2, 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, 5, 6, 7, 8. Recent studies have also demonstrated that besides taking part in chondrogenesis and osteogenesis, BMPs are also involved in embryonic skeleton formation 9, 10, 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 confirmed that BMPs, like other members of the TGF-β superfamily (i.e., TGF-β 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-β 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-βs or activins, which require formation of heteromeric kinase receptor complexes between receptor type I and II for signal transduction 15, 16, 17, 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 membrane 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].

Section snippets

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

Internalization of BMP

Fig. 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 +37°C. With continuous exposure to high concentrations of BMP, no statistically significant increase was found after 2 h. During incubation of cells with

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-β 19, 24, our results indicate that receptor-bound BMP also undergoes rapid

Acknowledgements

This study was financially supported by the University of Tampere and the Medical Research Fund of Tampere University Hospital.

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