Attenuation of Hypertrophy in Human MSCs via Treatment with a Retinoic Acid Receptor Inverse Agonist

Abstract

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient "chondrocytes" in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.

Keywords: BMS; chondrogenesis; hypertrophy; inverse agonist; mesenchymal stem cells; retinoic acid receptor.

Figure 1

Potential interactions between retinoid signaling and the Wnt/β-catenin pathway on the cellular level for growth plate chondrocytes during endochondral ossification. The inactive retinol is transported to the cell by retinoid binding protein and translocated into the cytoplasm by the transporter STRAT6, where it is transformed into the active retinoic acid (RA) [19,37,38]. The retinoic acid is translocated into the nucleus by the cellular retinoic acid binding protein II (CRABP II) [39,40]. Binding of retinoic acid (RA) to the RA receptor would activate gene expression of Wnt proteins, receptors, and coreceptors which leads to an increased Wnt/β-catenin signaling followed by hypertrophic conversion [41]. RBP, retinoid binding protein; STRAT6, ”stimulated by retinoic acid“, receptor/Vit. A transporter; RDH, retinol dehydrogenase; RALDH, retinaldehyde dehydrogenase; RAR, retinoic acid receptor; RXR retinoic X receptor; CRABP II, cellular retinoic acid binding protein; RARE, retinoic acid response element; GSK3, glycogen synthase kinase 3 CoA co-activator; LRP, lipoprotein receptor related protein; LEF/TCF, lymphoid enhancer binding factor/transcription factor, ↑, upregulation, ↓, downregulation.

Figure 2

Schematic demonstration of the inhibition of the RAR pathway by BMS. BMS is translocated into the nucleus by the cellular retinoic acid binding protein II (CRABP II) and binds to the RAR/RXR complex. Binding of the inverse agonist supports corepressor recruitment. The receptor complex subsequently inhibits target gene expression at the promoter area RARE. The consequently reduced expression of Wnts and Wnt receptors and coreceptors decreases hypertrophic differentiation [29,43,44]. RAR, retinoic acid receptor; RXR, retinoic X receptor; CRABP II, cellular retinoic acid binding protein; RARE, retinoic acid response element; CoA, co-activator; CoR, co-repressor; LRP, lipoprotein receptor related protein; LEF/TCF, lymphoid enhancer binding factor/transcription factor, ↓↓ reduction.

Figure 3

Effect of BMS and RA to the histological appearance of mesenchymal stem cells (MSC) aggregates on Day 28. First line ALP staining; second line DMMB staining; third line immunohistochemical collagen type II staining; fourth line immunohistochemical collagen type X staining. Scale bar = 500 μm. ALP, alkaline phosphatase; DMMB, dimethylmethylene blue; Coll II, collagen type II; Coll X, collagen type X; Chon, chondrogenic control group; Hyp, hypertrophic control group; Hyp BMS, hypertrophic group with late BMS treatment; BMS/Hyp, hypertrophic group with early BMS treatment; Hyp RA, hypertrophic group with late retinoic acid treatment; RA/Hyp, hypertrophic group with early retinoic acid treatment.

Figure 4

Effect of BMS on the number and size of hypertrophic cells. Image morphological determination of (A) number of hypertrophic cells relative to aggregate area; and (B) average cell size of hypertrophic cells in histological sections; (C) 40x magnification of the immunohistochemical collagen type II stained histological sections for illustration of hypertrophic chondrocytes under BMS treatment. n = 3. Whiskers represent standard deviations. Significant differences (* p < 0.05) are designated by asterisks. Scale bar = 25 μm. Chon, chondrogenic control group; Hyp, hypertrophic control group; Hyp BMS, hypertrophic group with late BMS treatment; BMS/Hyp, hypertrophic group with early BMS treatment.

Figure 5

Effect of BMS and RA on ALP activity and GAG content. ALP Enzyme activity is measured densitometrically in cell culture supernatants via conversion of an ALP substrate on Day 28 (A); GAG content of MSC pellets on Day 28 is normalized to DNA content (B). n = 3 MSC donor populations in groups obtaining RA resp. n = 6 populations in other groups. Whiskers represent standard deviations. Significant differences (* p < 0.05) are designated by asterisks. Chon, chondrogenic control group; Hyp, hypertrophic control group; Hyp BMS, hypertrophic group with late BMS treatment; BMS/Hyp, hypertrophic group with early BMS treatment; Hyp RA, hypertrophic group with late retinoic acid treatment; RA/Hyp, hypertrophic group with early retinoic acid treatment.

Figure 6

Effect of BMS and RA on gene expression. Quantitative PCR of the osteogenic marker collagen type I (A); the chondrogenic marker collagen type II (B); and the hypertrophic markers, MMP13 (C); and collagen type X (D), in MSC aggregates on Day 28. Relative gene expression normalized to Day 0 is given for n = 3 MSC donor populations in groups obtaining RA resp. n = 6 populations in other groups. Gene abbreviations are according to the NCBI database. Whiskers represent standard deviations. Chon, chondrogenic control group; Hyp, hypertrophic control group; Hyp BMS, hypertrophic group with late BMS treatment; BMS/Hyp, hypertrophic group with early BMS treatment; Hyp RA, hypertrophic group with late retinoic acid treatment; RA/Hyp, hypertrophic group with early retinoic acid treatment.