HDAC6 regulates dental mesenchymal stem cells and osteoclast differentiation

Bone is continuously undergoing resorption and remodeling to maintain bone volume and calcium homeostasis. Osteoblasts and osteoclasts play critical roles in regulating bone formation and resorption. Osteoblast cells are believed to derive from MSCs that originate in bone marrow [1]. MSCs are adult stem cells which can differentiate into chondrocytes, adipocytes, osteoblasts, myocytes, and hepatocytes [2, 3]. Osteoclasts are multinucleated cells differentiated from hematopoietic stem cells (HSCs) [4, 5]. Because the regulation of osteoclastogenesis and osteoblastogenesis are critical in tooth eruption and movements [6], many studies have been conducted to unveil the mechanism involved in bone cell differentiation. Broad-acting histone deacetylase (HDAC) inhibitor TAS has been reported to increase osteoblast proliferation and the transcriptional activity of Runx2 [7]. Another study demonstrated that HDAC inhibitor FR901228 decreased the maturation of osteoclasts [8]. However, there is still much to be understood about the specific role of individual HDACs in the regulation of dental bone resorption and regeneration.

Studies have shown that dental MSCs have multipotency for odontogenic, osteogenic, chondrogenic, and adipogenic potential under different culture conditions [9‐11]. Therefore, dental MSCs are a promising treatment approach for clinical applications in dental regeneration and craniofacial therapies. The features of dental MSCs are influenced by various factors including cytokines, cell passages, and oxygen concentration. In the last decade, our knowledge about molecular contributors to MSCs differentiation has also increased. Bone lineage commitment is reportedly regulated by the expression of transcription factor RUNX2, which further promotes expression of alkaline phosphatase (ALP), osterix (OSX), osteopontin (OPN), and osteocalcin (OCN) [12]. In addition, several transcription factors and signal pathways have been demonstrated to regulate MSCs differentiation [13, 14].

Osteoclasts are multinucleated cells differentiated from monocytes or macrophages that absorb bone matrix. Osteoclast differentiation is regulated by the tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor (NF)-κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) [15‐17]. Studies have suggested that TNF receptor-associated factor 6 (TRAF-6), NF-κB and c-fos are also essential molecules for osteoclast genesis [17]. In addition, genome-wide screening has provided insights into additional genes that are involved in the regulation of osteoclast differentiation.

Histone deacetylases (HDACs) are a group of conserved enzymes that remove acetyl groups from lysine side chains of both histones and nonhistone proteins. HDACs are reportedly involved in the regulation of bone formation and maintenance [18, 19]. HDAC6, a unique protein that belongs to class IIb of HDACs, can shuttle between the cytoplasm and nucleus. Previous studies have demonstrated that HDAC6 inhibitors accelerate osteoblast maturation and suppress osteoclast differentiation [20]. HDAC6-deficient mice develop a slightly enlarged tibia and increased bone mineral density [21]. The Rho–mDia2–HDAC6 pathway is reportedly involved with osteoclast maturation by controlling podosome patterning through microtubule acetylation in osteoclasts [22]. While these studies indicate a role for HDAC6 in bone growth and resorption, the role of HDAC6 in dental MSCs and osteoclast differentiation is still unclear. In this study, we demonstrate that HDAC6 knockdown in dental MSCs can promote osteoblast maturation. We further demonstrate that HDAC6 loss can inhibit osteoclast differentiation. Thus, our findings suggest that HDAC6 plays an important role in dental bone growth and maintenance.

HDAC6 inhibits odontogenic differentiation of dental MSCs.

Previous studies have suggested that the inhibition of HDAC6 could promote BMSCs differentiation. Dental MSCs are reportedly capable of differentiating into odontoblasts similar to BMSCs. Therefore, we designed experiments to explore the function of HDAC6 in dental MSCs differentiation. Dental MSCs were infected with lentivirus-carrying HDAC6 shRNAs to generate HDAC6 knockdown cell lines. The knockdown efficiency of two different HDAC6 shRNAs was confirmed by Western blot (Fig. 1a). As HDAC6 sh2 showed higher knockdown efficiency, we chose dental MSCs infected with HDAC sh2 for use in further experiments. When these cells were induced with bone formation medium, MSC/sh2 cells showed increased bone formation as demonstrated by ALP staining and activity on day 7 (Fig. 1b). Furthermore, HDAC6 knockdown also increased the formation of mineralized nodules after prolonged induction with bone formation medium for 21 days as demonstrated by ARS staining (Fig. 1c). By real-time RT-PCR, we examined mRNA expression of odontogenic marker genes at different time points after bone formation induction. Our results showed that HDAC6 knockdown significantly induced the expression of OSX (Fig. 1e), ALP (Fig. 1f), OCN (Fig. 1g), and OPN (Fig. 1h). Taken together, these results indicate that HDAC6 plays an important role in odontogenic differentiation of MSCs.

Rescue of HDAC6 inhibits odontogenic differentiation of dental MSCs.

To validate our finding from HDAC6 knockdown dental MSCs, we restored HDAC6 expression in MSCs expressing HDAC6 shRNA by infecting the cells with lentivirus-expressing Flag-HDAC6. Expression of HDAC6 was confirmed by Western blot (Fig. 2a). When HDAC6 sh2/Flag-HDAC6 cells were cultured with osteogenic medium, the cells showed similar bone formation capacity to control cells (scrsh/V), whereas HDAC6 sh2/V cells showed impaired bone formation capacity in ALP (Fig. 2b) and ARS assays (Fig. 2c). Similarly, the expression of OSX, ALP, OCN, and OPN as a result of HDAC6 knockdown was impaired after HDAC6 expression was restored (Fig. 2d–g), indicating that HDAC6 inhibits odontogenic differentiation.

Using dental MSCs to regenerate forms and functions of teeth is one of the important goals in dental tissue engineering, although additional fields of application are quickly emerging. Previous studies have discovered mechanisms involved in regulating odontogenic differentiation potential in dental MSCs [28, 29]. While dental MSCs are similar to BMSCs in their potential to differentiate into mineralized tissues, dental MSCs have more restricted differentiation than bone marrow cells have in vivo. It has been reported that dental MSCs are more committed to odontogenic than to osteogenic development in vitro [30]. Stem cells derived from dental tissue have been used for tissue engineering studies to assess their potential in pre-clinical application [31‐34]. Therefore, a better understanding of the molecular mechanisms underlying cell differentiation of dental MSCs is of significant interest in regenerative medicine.

Osteoclasts are highly specialized migratory cells that regulate bone resorption. They are critical for normal skeletal growth and the maintenance of bone integrity throughout life. Overactive osteoclasts may cause several bone diseases including Paget’s disease in bone, juvenile Paget’s disease, expansile skeletal hyperphosphatasia, and familial expansile osteolysis [35‐39]. Dental problems resulting from osteoclast activation have long been recognized. To improve our understanding and treatment of dental abnormalities and osteoclast diseases, it is critical to study mechanisms involved in regulation of osteoclasts differentiation.

HDACs can induce specific changes in gene expression by deacetylating both histone and non-histone proteins. HDAC inhibitors have been used in clinics for treatment of cancer, epilepsy, and bipolar disorder. In 1993, Iwami and Moriyama demonstrated that the HDAC inhibitor NaB could induce ALP expression in a MC3T3-E1 pre-osteoblast cell line [40]. Several studies suggest the HDAC inhibitor TSA has stimulatory effects on osteoblast cell lines, primary calvarial osteoblasts, and in calvarial organ cultures [7, 41]. TSA and NaB inhibit RANKL-mediated osteoclast differentiation from hematopoietic precursors [42]. Furthermore, several HDACs play important roles in bone development and physiology; HDAC1, HDAC3, HDAC6, and HDAC7 are involved with osteoblastogenesis through cooperation with, or inhibition of, Runx2 [43‐46]. There is still much to learn about the roles of HDACs in osteoclast differentiation. Interestingly, a study from 2011 showed that HDAC3 and HDAC7 play opposite roles in osteoclast differentiation [47]. Suppression of HDAC7 facilitated osteoclastogenesis and increased osteoclast size [48]. HDAC9 knockout mice had elevated osteoclast numbers and bone resorption indices [49]. Class III HDAC Sirt1 also inhibited osteoclast differentiation in a conditional deletion Sirt1 mouse model [50].

As a member of the class II HDAC family, HDAC6 predominantly localizes in the cytoplasm. It plays an important role in regulating cell motility by deacetylating a-tubulin and cortactin [51, 52]. HDAC6 also modulates Hsp90-dependent activation of the glucocorticoid receptor through deacetylating Hsp90 [53, 54]. Loss of HDAC6 increased the formation of ossified bone in TDII embryos, but it did not lead to significantly different bone mineral densities at later ages [55, 56]. Specific inhibition of HDAC6 with Tubastatin or AC1215 increased osteoblast formation and osteoclast inhibition [20]. Taking into account previous published results, we have shown that HDAC6 knockdown promotes dental MSCs differentiation by using shRNA. By employing a small interfering RNA technique, we also knocked down the expression of HDAC6 in murine osteoclast precursor cells line RAW 264.7. The knockdown of HDAC6 in RAW 264.7 cells inhibited osteoclasts formation under RANKL stimulation. However, it is still not clear whether HDAC6 could regulate osteoclast precursor proliferation. Although further studies are required to understand the detailed molecular mechanisms by which HDAC6 regulates the expression of osteoclast marker genes, our results shed light on the role of HDAC6 in dental MSCs and osteoclasts differentiation. Together with previous findings, our study suggests that HDAC6 is a potential regulator for bone growth and maintenance.

We expect that HDAC6 knockdown will lead to inhibition of osteoclastogenesis and the promotion of dental MSCs odontogenic differentiation. Our findings give new insight into the role of HDACs in tooth development and disease and suggest HDAC6 as a novel therapeutic target for disease treatment and drug development. HDAC6 knockout mice appear to be healthy, and highly specific HDAC inhibitors appear to be well tolerated in clinical trials. The inhibition of HDAC6 may offer health advantages under some dental conditions. In future studies, it will be important to determine how HDAC6 is involved with networks regulating dental cell differentiation.