Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists

Abstract

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.

Figures

Figure 1

RAR agonists block chondrogenesis and intramuscular rBMP-2-driven HO. (a) E11.5 mouse limb mesenchymal cells in micromass cultures were treated with increasing doses of RA or RARγ agonist NRX204647 and were stained with Alcian blue on day 8. (b) Similar micromass cultures prepared with E11.5 RARγ-null or wild type (WT) limb mesenchymal cells were treated with RA (100nM) for 8 days. RA inhibited chondrogenesis in WT, but not mutant cultures. (c) Micromass cultures prepared with dual RARα/RARβ-deficient or WT limb mesenchymal cells were treated with RA as above. In this case, RA inhibited chondrogenesis in both cultures. (d) HO was induced by implantation of rBMP-2-filled collagen sponge into a microsurgically-created pocket inside the calf muscles and the mice were then treated with vehicle, RA (12 mg per kg body weight per day) or NRX204647 (1.2 mg per kg body weight per day) by gavage. Note the large round mineralized HO masses visible by μCT in vehicle- receiving control mice, their significant decrease in RA-treated mice and their absence in NRX204647-treated mice. Ectopic tissues were sectioned and examined by Masson trichrome (MT) and Alcian blue (AB) staining, immuno-fluorescence detection of myosin heavy chain (MHC: red) and osteocalcin (OC: green), and TRAP staining. Size bar for μCT: 5 mm; and for histology: 200 μm. (e) HO was induced by subcutaneous implantation of rBMP-2/Matrigel mixture. Treatment condition and analysis of ectopic tissue were done exactly as above. Bar inμCT panels, 5.0 mm; bar in AB panels, 2.5 mm.

Figure 2

Effectiveness of different retinoids against HO. (a) Chemical structures of synthetic RARγ agonists and natural 13-cis-RA. (b) Suppression of HO by different retinoids was evaluated by measuring bone volume/total volume (BV/TV) in ectopic masses in control versus retinoid-treated mice by μCT on day 12. Values from control groups were set at 100% in each experiment and used to calculate relative values in experimental groups. Each group had a minimum of 8 samples, and data are presented as means average ± s.e.m. (* p< 0.01 vs control). (c) Macro views (top), soft x-ray radiograms (middle) and H&E stained histological sections (bottom) of ectopic masses collected from vehicle-treated control and CD1530-treated mice. Bar in top and middle panels, 1.0 cm; and bar in bottom panel, 100 μm. (d) Selectivity of RARγ agonist action. Subcutaneous HO was triggered in WT, heterozygous RARγ+/−− (HT) and RARγ-null (KO) mice, and mice were treated with RARγ agonist CD1530 (4.0 mg per kg body weight per day) or vehicle for 12 days. * p< 0.01 vs control. (e) Evaluation of rebound effects. Mice implanted with rBMP-2/Matrigel mixture subcutaneously were treated with vehicle, CD1530 or RARα agonist NRX195183 for 10 days. Treatment was stopped, and HO was evaluated byμCT at later time points. (f) Window of opportunity tests. Mice implanted as above were left untreated for up to day 6 and were then treated with CD1530 or NRX195183 until day 12.

Figure 3

RARγ agonists block FOP-like HO formation. (a) ADTC5 cells expressing the strong constitutive-active ALK2Q207D or control empty vector were transfected with the BMP signaling reporter Id1-luc and then treated with 0, 10, 30 and 100 nM CD1530. Reporter activity was normalized to phRG-TK. The strong reporter activity in ALK2Q207D expressing cells was counteracted by RARγ agonist treatment. (b) ALK2Q207D transgenic mouse model of FOP-like HO. Consecutive soft x-ray images of the same Ad-Cre/cardiotoxin-injected mice taken at P7, P21 and P35 showing that massive HO was present in vehicle-treated mice (double arrowheads), but was dramatically reduced in companion CD1530-treated mice (4.0 mg per kg body weight per day). Bar, 1.0 cm. (c) μCT images showing that massive HO was present in vehicle-treated Ad-Cre/cardiotoxin-injected ALK2Q207D transgenic mice (double arrowheads), but was essentially absent in companion CD1530-treated mice. Bar, 1.0 cm. (d) Bright field (BF) and fluorescent images showing that ectopic Alizarin complexon-positive skeletal tissue (AR) had formed in control Ad-Cre/cardiotoxin-injected ALK2Q207D transgenic mice receiving vehicle (middle lower panel), but not in those receiving CD1530 (right lower panel). Positive GFP fluorescence verified that Ad-Cre had activated transgene expression in all mice. No GFP and AR fluorescence was seen in mice that had not been injected with Ad-Cre/cardiotoxin (left panels). Bar, 5.0 mm.

Figure 4

Mechanisms of RARγ agonist action. (a) Id1-luc reporter activity in ATDC5 cells. (b) Immunoblots showing that: (i) phosphorylated Smad1/5/8 protein levels were transiently but markedly increased in control (DMSO) ATDC5 cells treated with rBMP-2, but much less in cells treated with CD1530 (top panel); and (ii) overall Smad1 levels did not change in control cells over time, but decreased progressively and markedly in CD1530-treated cells (bottom panel). Membranes were re-blotted with α-tubulin antibodies for normalization. (c) Dual Smad1 and β-actin immunofluorescence staining and nuclear DAPI staining showing that Smad1 was largely cytoplasmic in control cells and relocated to the nucleus during rBMP-2 treatment, but there was minimal detectable Smad1 signal in cells co-treated with CD1530 in either cytoplasm or nucleus. Smad1 is in red, β-actin is in green, and nuclei are in blue. (d) Immunoblots similar to those in (b) showing that the overall levels of Smad1, Smad4 and Smad5 were all decreased by CD1530 treatment with or without rBMP-2 treatment. (e) Immunoblots showing that the decreases in Smad1 levels elicited by CD1530 treatment were significantly counteracted by co-treatment with proteasome inhibitors AW9155 or PI-108.

Figure 5

RARγ agonists reprogram the differentiation potentials of skeletal progenitor cells. (a- b) ATDC5 cells were grown in the presence of vehicle (control) or 1 μM CD1530 for 2 days, rinsed, re-plated and then grown for an additional 7 days with or without 100 ng ml−1 rBMP-2. Note that control cells responded well to rBMP-2 and underwent chondrogenic differentiation revealed by strong Alcian blue (a) and alkaline phosphatase (b) staining, but the CD1530 pre-treated cells did not and failed to stain. (c) Similar experiments with GFP-expressing mouse bone marrow-derived MSCs show that the cells underwent differentiation upon rBMP-2 treatment (top row), but CD1530 pre-treated MSCs did not and failed to stain with alizarin red (bottom row). (d) Immunoblots showing the steady state levels of Smad1 and Smad4 in MSCs treated without (−) or with (+) 1 μM CD1530 for 12 hr. (e) Id1-luc reporter activity in control MSCs treated with or without rBMP-2 in the presence or absence of CD1530. (f) Control and CD1530-pretreated GFP-expressing MSCs were mixed with rBMP-2/Matrigel and implanted in nude mice subcutaneously, and ectopic tissue masses were analyzed by μCT, H&E staining, osteocalcin (OC) immunostaining and GFP fluorescence signal. Merged images of GFP and OC images are shown at the bottom. Bar in top panel, 5.0 mm; bar in second panel, 250 μm; and bar in third panel, 150 μm.