The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies

Abstract

Mesenchymal stem cells (MSCs) have been identified in many adult tissues. MSCs can regenerate through cell division or differentiate into adipocytes, osteoblasts and chondrocytes. As a result, MSCs have become an important source of cells in tissue engineering and regenerative medicine for bone tissue and cartilage. Several epigenetic factors are believed to play a role in MSCs differentiation. Among these, microRNA (miRNA) regulation is involved in the fine modulation of gene expression during osteogenic/chondrogenic differentiation. It has been reported that miRNAs are involved in bone homeostasis by modulating osteoblast gene expression. In addition, countless evidence has demonstrated that miRNAs dysregulation is involved in the development of osteoporosis and bone fractures. The deregulation of miRNAs expression has also been associated with several malignancies including bone cancer. In this context, bone-associated circulating miRNAs may be useful biomarkers for determining the predisposition, onset and development of osteoporosis, as well as in clinical applications to improve the diagnosis, follow-up and treatment of cancer and metastases. Overall, this review will provide an overview of how miRNAs activities participate in osteogenic/chondrogenic differentiation, while addressing the role of miRNA regulatory effects on target genes. Finally, the role of miRNAs in pathologies and therapies will be presented.

Keywords: MSC differentiation; disease; epigenetics; microRNAs; tumour.

Figure 1

MiRNA biogenesis. MiRNAs are transcribed by RNA polymerases II (Pol II) in the form of a first precursor called primary miRNA (pri-miRNA). The pri-miRNA is converted into the precursor miRNA (pre-miRNA) via the cutting activity of the Drosha enzyme, a nuclear endoribonuclease III. Pre-miRNAs are exported into the cytoplasm as a result of the action of Ran-GTP and Exportin-5, a nuclear export factor. In the cytoplasm, Dicer clivates the pre-miRNA into a double stranded miRNA (miRNA duplex) of about 18-22 nt. The mature miRNA strand is incorporated into the RNA- induced silencing complex (RISC), which guides the miRNAs to the 3′UTR of its target.

Figure 2

SOX9 regulation during chondrogenesis. A) In chondrogenesis, SOX9 is negatively regulated by WNT, NOTCH and retinoic acid pathways. Otherwise, TGF-β, BMP, FGF, IHH, PTHrP, HIF-1 pathways positively regulate SOX9 expression. SOX9 induces COL2A1, COL9A1, COL11A2, COMP and Agc1 expression and inhibits RUNX expression, which in turn activates COL10a1, MMP13, VEGF-A and FGF-18 genes. B) Endochondral ossification is regulated by transcription factors. SOX9 promotes the commitment of mesenchymal stem cells into chondrocytes; SOX5/6/9 promotes chondrocytes differentiation; during chondrocyte hypertrophy, SOX5/6/9 expression decreases and RUNX and OSX expression is activated to promote bone formation.

Figure 3

TGF-β and BMPs signaling pathways in osteogenesis. TGF-βs and BMPs bind to the extracellular domains of specific receptors and require Sma and Mad related (SMAD) proteins for signal transduction within the cells. TGF-βs and BMPs work through heterodimeric receptors consisting of type I and type II kinase receptors. TβRI/ALK5 is the type I receptor for TGF-βs, and BMPR1A/ALK3, BMPR1B/ALK6 and AcvR1/ALK2 are type I receptors for BMPs. TβRII/TGFBR2 and BMPR2 are the type II kinase receptor for TGF-βs and BMPs, respectively. Upon binding of the ligand to the receptor, the receptor forms homodimeric complexes, resulting in one subunit phosphorylating its partner subunit on the serine/threonine residues. This starts a cascade of events involving SMAD protein phosphorylation. The TGF-β pathway requires SMAD2 and SMAD3, whereas BMP signaling is dependent on SMAD1, 5 and 8. Phosphorylated receptor-regulated SMADs (R-SMAD) react with SMAD4 to create a heterocomplex. This complex, consisting of R-SMAD and common-mediator SMAD (Co-SMAD), enters the nucleus and controls transcription by binding to target gene promoters. Phosphorylated R-SMAD (R-SMAD-Pi) interacts with SMAD4 protein, then translocates into the cell nucleus where AMP response element binding protein (CREB)-binding protein (CBP) and P300 coactivators are recruited which regulate gene targets. SMAD2/3-Pi in the nucleus and SMAD4-unlinked recruit HDAC4 and HDAC5 by inhibiting RUNX2 and OSX expression. In the Non-Smad-dependent signaling pathway, TGF-β activates kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) to initiate the MKKs (MAPK pathway member-encoding genes kinases)-p38 MAPK or -Erk (extracellular signal regulated kinase) signaling cascades. Interaction between BMPs and their receptors result in comparable events triggered by TGF‐β. In BMP signaling, R-SMAD (SMAD1/5/8) bind SMAD4 to translocate into the nucleus, where they induce RUNX2 expression. Similarly, Non-Smad-dependent BMP-stimulated pathway induces DLX5, RUNX2, and OSX expression and leads to osteoblast and osteocyte differentiation. Latent TGF-beta binding proteins (LTBPs) bind TGF-β and prevent its interaction with TβRI and TβRII receptors; otherwise, Noggin, Chordin, Gremlin and Follistatin bind to BMP proteins and block their signaling cascades. In TGF- β signaling, SMAD7 inhibits SMAD2/3 translocation into the nucleus, and, alongside Smurf2 induces R-SMAD proteasome-mediated degradation. Similarly, in BMP signaling, SMAD6/7 inhibit SMAD1/5/8 translocation into the nucleus and, alongside Smurf1/2 induces SMAD1/5/8 and RUNX2 proteasome-mediated degradation. In turn, SMAD6 expression is regulated by RUNX2 in a negative feedback loop. ARKADIA protein positively regulates osteoblast differentiation by binding SMAD6/7 proteins.