© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs246934. doi:10.1242/jcs.246934 RESEARCH ARTICLE BMPR1A is necessary for chondrogenesis and osteogenesis, whereas BMPR1B prevents hypertrophic differentiation Tanja Mang1,2, Kerstin Kleinschmidt-Doerr1, Frank Ploeger3, Andreas Schoenemann4, Sven Lindemann1 and Anne Gigout1,* ABSTRACT essential for osteogenesis and bone formation during this process BMP2 stimulates bone formation and signals preferably through BMP (Bandyopadhyay et al., 2006; McBride et al., 2014; Yang et al., 2013). – receptor (BMPR) 1A, whereas GDF5 is a cartilage inducer and signals Similarly, during bone fracture healing where a similar mechanism – preferably through BMPR1B. Consequently, BMPR1A and BMPR1B are takes place conditional deletion of Bmp2 in mesenchymal believed to be involved in bone and cartilage formation, respectively. progenitors or osteoprogenitors prevents fracture healing (Mi et al., However, their function is not yet fully clarified. In this study, GDF5 2013; Tsuji et al., 2006). In vitro, BMP2 provokes an induction of mutants with a decreased affinity for BMPR1A were generated. These alkaline phosphatase (ALP) activity, osteocalcin expression and matrix mutants, and wild-type GDF5 and BMP2, were tested for their ability to mineralization in pluripotent mesenchymal progenitor cells (Cheng induce dimerization of BMPR1A or BMPR1B with BMPR2, and for their et al., 2003), and also stimulates chondrogenesis or adipogenesis (Date chondrogenic, hypertrophic and osteogenic properties in chondrocytes, et al., 2004). Finally, BMP2 has been shown to promote bone repair in in the multipotent mesenchymal precursor cell line C3H10T1/2 and the animal models (Kleinschmidt et al., 2013; Wulsten et al., 2011) and in human osteosarcoma cell line Saos-2. Mutants with the lowest potency a clinical setting (Ronga et al., 2013). for inducing BMPR1A–BMPR2 dimerization exhibited minimal Growth differentiation factor 5 (GDF5; also named BMP14 or chondrogenic and osteogenic activities, indicating that BMPR1A is CDMP1) is also expressed in the developing limb at sites where joint necessary for chondrogenic and osteogenic differentiation. BMP2, GDF5 cavitation occurs, and null mutation of Gdf5 in mice disrupts the and the GDF5 R399E mutant stimulated expression of chondrogenic and formation of 30% of the synovial joints (Storm and Kingsley, 1996). hypertrophy markers in C3H10T1/2 cells and chondrocytes. However, Several gene expression or deletion studies strongly indicate that GDF5 R399E, which induces the dimerization of BMPR1B and BMPR2 GDF5 (together with GDF6 and GDF7) provides a specialized more potently than GDF5 or BMP2, displayed reduced hypertrophic function for joint morphogenesis (Settle et al., 2003; Storm and activity. Therefore, we postulate that stronger BMPR1B signaling, Kingsley, 1999). In vitro, GDF5 promotes chondrogenic compared to BMPR1A signaling, prevents chondrocyte hypertrophy differentiation and chondrocyte hypertrophy (Coleman et al., 2013; and acts as a cartilage stabilizer during joint morphogenesis. Erlacher et al., 1998). Subcutaneous implantation of collagen or collagen/hyaluronate loaded with GDF5 in the rat results in cartilage This article has an associated First Person interview with the first author formation followed by bone formation (Erlacher et al., 1998; Spiro of the paper. et al., 2000). In bone defect models, GDF5 caused delayed tissue mineralization compared to BMP2 and the presence of cartilage tissue KEY WORDS: GDF5, BMP2, M1673, Chondrogenesis, Hypertrophy, in the defect (Kleinschmidt et al., 2013; Wulsten et al., 2011). Finally, Osteogenesis intra-articular injections of GDF5 stimulated cartilage repair in a rat osteoarthritis model (Parrish et al., 2017). In conclusion, both BMP2 INTRODUCTION and GDF5 promote cartilage and bone formation, but BMP2 more Bone morphogenetic proteins (BMPs) play a role in a multitude of strongly promotes osteogenesis and bone formation than does GDF5. processes during embryonic development, including skeletal BMP2 and GDF5 elicit their effects through two types of serine/ development. Most skeletal elements are formed by endochondral threonine kinase transmembrane receptors; type I and type II ossification, which is initiated by mesenchymal cell condensation. receptors. There are three type II and seven type I receptors that Under the influence of several BMPs, these cells differentiate into interact with BMPs, and the association of a type I with a type II chondrocytes that produce cartilaginous tissue, and further differentiate receptor is required for the formation of an active signaling complex. into hypertrophic chondrocytes. Afterward, vascularization takes place BMP2 and GDF5 are both known to interact with all three type II concomitantly with replacement of cartilage into bone, driven by receptors (BMPR2, ACVR1 and ACVR2) together with the type I osteoblasts and osteoclasts (Katagiri and Watabe, 2016; Salazar et al., receptors BMPR1A or BMPR1B. However, BMP2 and GDF5 2016). Several conditional deletion studies have shown that BMP2 is preferentially interact with BMPR1A and BMPR1B, respectively (Heinecke et al., 2009; Nishitoh et al., 1996). In accordance with the 1Osteoarthritis Research, Merck KGaA, 64293 Darmstadt, Germany. 2Institute for role of BMPs in skeletal development, the receptors Bmpr1a and Organic Chemistry and Biochemistry, Technische Universität, 64289 Darmstadt, Bmpr1b are expressed in the developing limbs (Baur et al., 2000; Germany. 3Biopharm GmbH, 69214 Eppelheim, Germany. 4Discovery Dewulf et al., 1995; Zou et al., 1997). Several studies have aimed to Pharmacology, Merck KGaA, 64293 Darmstadt, Germany. decipher the role of BMPR1A and BMPR1B in skeletal development. *Author for correspondence ([email protected]) In vivo, the use of loss-of-function or gain-of-function mutations revealed some redundancy in the role of these two BMP type I T.M., 0000-0002-6497-3034; A.G., 0000-0003-1399-7957 receptors (Kobayashi et al., 2005; Yoon et al., 2005). In vitro, Handling Editor: John Heath contradictory results have been published. For instance, it has been Received 26 March 2020; Accepted 21 July 2020 shown that constitutively active forms of BMPR1A and BMPR1B Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs246934. doi:10.1242/jcs.246934 activate chondrogenesis in ADTC5 cells (Fujii et al., 1999), whereas BMP2, GDF5, W417R and W417F. Similar to the Kd values, the forced expression of BMPR1A, but not BMPR1B, stimulates ratio of EC50 values were calculated. The same selectivity profile osteogenesis and chondrogenesis in C3H10T1/2 cells (Kaps et al., was obtained; BMP2 was more selective for BMPR1A than 2004). BMPR1B and the selectivity was shifted toward BMPR1B with In conclusion, the respective role of BMPR1A and BMPR1B in GDF5, R399E, W417F and W417R. chondrogenesis and osteogenesis is not yet fully clear. In this study, we tackled this question from a different angle. Although genetic BMPR expression, Nog sensitivity and Nog expression manipulations are extremely helpful, the results should be interpreted To ensure that the cells selected to study chondrogenesis and with caution. Silencing the expression of one protein can be osteogenesis are relevant, we verified that they express the receptors compensated by the expression of other similar proteins (Rossi we intend to study. The C3H10T1/2 cells, Saos-2 cells and porcine et al., 2015), while overexpression induces non-physiological protein chondrocytes all express Bmpr1a/BMPR1A, Bmpr1b/BMPR1B and expression levels and might generate results that do not accurately Bmpr2/BMPR2 (Fig. S1). In addition, because the proteins we use reflect the biology. As an alternative, we generated several mutants of in this work derive from the human GDF5 sequence and were tested GDF5 that differentially activate BMPR1A or BMPR1B. These on human receptors for the affinity or dimerization measurements, mutants were tested together with wild-type GDF5 and BMP2 for we also evaluated the percentage identity between human, porcine their chondrogenic, hypertrophic and osteogenic properties in the or mouse BMPRs (see Table S1). For all sequences, a high identity multipotent mesenchymal precursor cell line C3H10T1/2, primary was found (>96%) and variations within the sequence are limited to chondrocytes and the human osteosarcoma cell line Saos-2. regions that are not involved into ligand binding (Kotzsch et al., 2009; Weber et al., 2007; Mace et al., 2006). RESULTS Finally, we also evaluated the noggin (Nog) sensitivity of the Binding affinity of BMP2, GDF5 and the GDF5 mutants as GDF5 mutants. Nog is an inhibitor of BMPs that can be produced by measured by surface plasmon resonance chondrocytes or C3H10T1/2 cells (Kameda et al., 2000; Zehentner The binding affinities of BMP2, GDF5 and the three GDF5 mutants et al., 2002). It was previously shown that Noggin inhibits GDF5, for BMPR2, BMPR1A and BMPR1B were measured by surface and that specific mutations of GDF5 can suppress Noggin plasmon resonance (SPR) (Table 1). The affinity measurements of inhibition. (Seemann et al., 2009). We found cells that stimulated BMP2, GDF5, R399E and W417F with BMPR2 resulted in Kd with GDF5, R399E and BMP2 were strongly sensitive to Nog values in the 2-digit nM range, while no binding of W417R could be treatment but those stimulated with W417F and W417R were less detected. The Kd values obtained for BMPR1B were in the 2- to affected