Mesenchymal stem cells (MSCs) are capable of differentiating into osteoblasts and adipocytes, and the dysregulation of MSC lineage specification may lead to the imbalance between bone mass and fat tissue, which finally result in osteoporosis. Our previous study has shown the critical role of KDM4B in promoting osteogenic differentiation and reducing adipogenic differentiation of MSCs in vitro, but the in vivo function of KDM4B remains to be investigated. In this study, Global deletion of Kdm4b and deletion of Kdm4b in mesenchymal progenitors, but not in mature osteoblasts, enhanced age-related bone loss and adipose accumulation in mouse bone marrow. Deletion of Kdm4b in mesenchymal progenitors also promoted bone loss and adipose accumulation induced by estrogen deficiency respectively. Restoration of KDM4B successfully reinstated ALP activity, mineralization and expression of osteogenic-related genes, and inhibited adipogenesis of BMSCs from Prx1-Cre;Kdm4bfl/fl mice. Furthermore, Kdm4b was required for the self-renewal of mouse MSCs, as determined by colony formation assay and serial transplantation assay. Collectively, Kdm4b was required for bone homeostasis by promoting osteogenic differentiation and reducing adipogenic differentiation of MSCs in vivo. Kdm4b was also required for self-renewal of MSCs. Targeting KDM4B may facilitate the prevention and therapy of osteoporosis, and also be used in MSC-based bone regeneration. In addition, we reported an efficient ex vivo culture protocol to derive functional MSCs from human ESCs by inhibition of NFκB. We found inhibition of NFκB promoted loss of the pluripotent markers during differentiation of human ESCs, and increased expression of mesenchymal lineage markers. Microarray analysis revealed that the genes regulated by inhibition of NFκB signaling are associated with developmental process and cell differentiation during human ESC differentiation. Finally, the purified MSCs showed multipotency in vitro. Our data provides key insights into the role of NFκB in mesenchymal lineage specification of human ESCs and provide a novel method for generation of MSCs.

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp (DP) and periodontal ligament (PDL) tissues using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) and compared their capacity to undergo multilineage differentiation. Fluorescence-Activated Cell Sorting (FACS) revealed that 27.3% of DPCs were CD51+/CD140α+, 10.6% were CD271+, and 0.3% were STRO-1+/CD146+; whereas 24% of PDLCs were CD51+/CD140α+, 0.8% were CD271+, and 2.4% were STRO-1+/CD146+. Sorted cell populations were further assessed for their multipotent properties by inducing odonto/osteogenic, chondrogenic, and adipogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into odonto/osteogenic and chondrogenic lineages but with varying degrees. CD271+ DMSCs demonstrated the greatest odonto/osteogenic potential with strong induction of odonto/osteogenic and periodontal markers such as DLX5, RUNX2, BGLAP, DMP1, DSPP, and PLAP-1. Based on these results, the role of CD271 in odontogenic differentiation was further evaluated. CD271 is one of the receptors which Nerve Growth Factor (NGF) exerts its biological effects through. Although NGF is known for its role in the the development and maintenance of the nervous system, it’s capability to promote odontogenic differentiation has made it an interesting candidate for its role as a mineralizing agent in regenerative therapies. We found that exogenous treatment of NGF further increased odonotogenic potential of CD271+ DMSCs from DP with induction of odontogenic markers DLX5, RUNX2, and BGLAP, and increased MAPK, AKT, MTOR, and NF-B signaling pathways in a time dependent manner. Furthermore, knockdown of tropomyosin trkA tyrosine kinase receptor (trkANGFR) and low affinity nerve growth factor receptor (p75NTR/CD271/LNGFR) decreased odontogenic differentiation of CD271+ DMSCs. Our study provides important insights into the use of DMSCs and growth factors for regenerative therapies in dentistry, and improves our understanding of the molecular mechanisms involved in odontogenic differentiation

Development of chemoresistance, invasive growth, and metastasis remain key challenges in head and neck squamous cell carcinoma (HNSCC) therapy. Recent studies revealed that an activated hepatocyte growth factor receptor (MET) is frequently overexpressed and highly associated with HNSCC invasion and metastasis. Also, autophagy, a highly conservative intracellular recycling system, has shown to play a primary role in cancer cells to attenuate cytotoxicity of chemoreagents in many hematopoietic and solid cancers. However, little is known about the epigenetic regulation of the MET signaling pathway or autophagy induction and whether it plays a role in promoting HNSCC invasion and metastasis or development of resistance to therapy. In our study, we found that histone deacetylase 6 (HDAC6) is a key epigenetic regulator of autophagy that promotes chemoresistance in HNSCC against the proteasome inhibitor, Bortezomib. The depletion of HDAC6 inhibited autophagy activation and enhanced Bortezomib-induced apoptosis in HNSCC cells. Mechanistically, we revealed that HDAC6 mediated activation of autophagy by modulating activation of protein kinases such as unc-51 like autophagy activating kinase 1 (ULK1) to promote clearing of large quantities of cytotoxic, unfolded protein aggregates induced by the Bortezomib. In addition, we found histone demethylases KDM6B plays an important role in acquiring cisplatin resistance in HNSCC and in unraveling the mechanism associated with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Interestingly, we also identified KDM6B as an essential epigenetic regulator of MET-driven HNSCC metastasis. KDM6B was highly expressed in both the growth factor-induced HNSCC cells and in cells overexpressing the oncogenic translocated promoter region MET (TPR-MET). KDM6B knockdown significantly decreased the HNSCC invasion and metastasis by regulating the expression of ETS proto-oncogene 1 (ETS1) and the high mobility group AT-Hook 2 (HMGA2) genes, known as the drivers of metastasis. Mechanistically, KDM6B facilitated the binding of the transcription factor ELK1, a downstream target of c-MET signaling pathway, to the promoters of ETS1 and HMGA2. In conclusion, our study provides insight into the epigenetic regulation of HNSCC chemoresistance, invasion, and metastasis and suggests that HDAC6 and KDM6B could be an important therapeutic target to improve chemotherapeutic efficacy and to decrease the tumor burden of HNSCC patients.

The intricate anatomy of the primary tumor, the occurrence of late-stage diagnosis, combined with the aggressive nature of head and neck squamous cell carcinoma (HNSCC) has made this disease difficult to treat. The global rate of HNSCC continues to rise, accounting up to 25% of all new cancer cases. But despite advances in treatment, the five year survival rate has remained stagnant for decades. Therefore a clearer understanding of the formation and progression of HNSCC is essential to the development of better therapeutic approaches to prevent and treat HNSCC patients. Recent studies have identified yes associated protein (YAP) as a potential target. YAP is a highly conserved transcriptional coactivator and has been found amplified as well as its expression upregulated and active in HNSCCs. In this study we hypothesize that YAP plays a key role in the development and progression of HNSCC. Our results show that activated YAP can transform and induce epithelial to mesenchymal transition (EMT) of our immortalized but nontransformed oral keratinocyte cell line OKF6 as seen by its ability to increase proliferation, saturation density, invasion, and induce anchorage independent growth. Moreover activated YAP potently enhances tumor formation and growth in vivo. YAP knockdown in HNSCC cell lines corroborated our initial findings where YAP knockdown could inhibit proliferation and invasion of HNSCC. We also discovered that YAP abundance correlated with tumor stage and lymph node metastasis in human HNSCC tissue samples. More recent studies have identified YAP regulation in response to extracellular cues. Therefore we further pursued our findings to determine whether YAP could mediate HGF induced cancer characteristics. We found that YAP knockdown could inhibit HGF induced proliferation and invasion of HNSCC, and by further microarray analysis, identified new potential targets and pathways by which YAP could direct cancer development and progression. Taken together our study not only suggests that YAP may play an important role in HNSCC, but also mediate response upon HGF stimulation to provide us with greater insight into the molecular regulation of HNSCC.

Tumor necrosis factor- alpha (TNF- α) is a major inflammatory cytokine that stimulates apoptotic signaling pathway and activates the transcription factor nuclear factor kappa B (NF- κB). Its contribution of apoptosis and rate of differentiation in regulating osteoblasts remains controversial. Recently, human mesenchymal stem cells were demonstrated in dental tissues. Human dental stem cells are also multipotent and can be induced to differentiate into different cell lineages. These cells are definitely a key part of achieving the promise of tissue and bone regeneration, along with bone marrow stem cells. In this research study, we wanted to see the effect of TNF- α on odontogenic differentiation of dental stem cells. We treated two different dental stem cell (DSCs) lines - dental pulp stem cells (DPSCs), and apical papilla (SCAPs) with 1ng/mL of TNF- α in different time points. Within 7 days, we could see an early alkaline phosphatase (ALP) expression and activity. In addition, ALP expression and activity were higher with treatment at 1ng/ml. Enhanced matrix mineralization was also observed with Alizarin Red Staining (ARS) after 14 days, and the mineralization was stronger with lower TNF- α concentration treatments. Furthermore, we investigated the effect of TNF-α on transcription factors, RUNX2 and OSX, two critical factors in osteogenic and odontogenic differentiation. The results showed that TNF- α induced the RUNX2 expression in both dental stem cells at different time points (4 hours and 24 hours). However, we observed a decrease in the expression of OSX. In this study, we demonstrated that TNF- α (at a lower concentration) could enhance odontogenic differentiation in dental stem cells. The amount of exposure of TNF- α might be a critical factor in determining its effects on odontoblast lineage commitment of dental stem cells.

DPSCs, also known as, dental pulp stem cells, were the first human dental mesenchymal stem cells (MSCs) to be identified from pulp tissues. DPSCs and their classification as mesenchymal stem cells is an attractive target for clinical applications in dentistry. DPSCs have been shown to differentiate into odontoblasts/osteoblasts, chondrocytes, and adipocytes. While studying a rare genetic disorder, Fan et al was able to uncover BCOR, a complex in which mutation could have intrinsic effect on the function of MSCs from the root apical papilla. One of the members of the BCOR complex is polycomb group ring finger 1 (PCGF1). PCGF1 is also a member of the polycomb group repressive complex 1 (PRC1). Due to its membership in repressive complexes, BCOR and PRC1, and its role in development, in this study we decided to investigate the role of PCGF1 in DPSC odontogenic differentiation. In order to evaluate the role of PCGF1 in DPSCs, we used small interfering RNA to silence the PCGF1 gene and observe the changes in our DPSC population. We found that after PCGF1 knockdown and treatment with odontogenic/osteogenic inducing media, mineralized nodule formation and odontogenic potential decreased, suggesting that PCGF1 plays a role in the odontogenic lineage commitment of DPSCs. We also found that mechanistically, PCGF1 blocks inhibition of developmental genes MSX1/MSX2 and DLX2/DLX5 in order to facilitate the odontogenic lineage commitment of DPSCs. Taken together, this study might shed light on the potential therapeutic implications of exploiting this pathway in DPSCs.

The human skeleton undergoes continuous bone remodeling, a process relying on orchestrated balance between the actions of osteoblasts and osteoclasts. Complex molecular signaling networks govern the differentiation and functions of mesenchymal stem cell (MSC)-derived osteoblasts and monocyte macrophage-derived osteoclasts, as local factors, immune cytokines and systemic hormones exert their regulatory effects. Moreover, the lineage decisions of MSCs to choose osteogenesis over adipogenesis is critical for maintenance of bone mass. MSC cell fate is determined by epigenetic regulations of various lineage-specific genes and genes promoting `stemness'. In most bone pathologies especially osteoporosis, the balance in bone remodeling and in MSC lineage decisions become disrupted. To explore the molecular regulation of bone cells, we evaluated the effect of Wnt4 on bone loss associated with osteoporosis and skeletal aging. We generated transgenic mice overexpressing Wnt4 in osteoblasts, and discovered that Wnt4 signaling could attenuate bone loss and suppress inflammation in models of osteoporosis, inflammatory and age-related bone loss. Mechanistically, non-canonical Wnt4 signaling could attenuate Nf-κb signaling by competitive sequestering of transforming growth factor associated kinase 1 (Tak1) in bone marrow macrophages. Furthermore, Wnt4 recombinant protein injection effectively prevented and reversed bone loss induced by estrogen-deficiency. Hence, non-canonical Wnt4 signaling could not only promote bone formation, but also inhibit bone resorption and inflammation in marrow microenvironment by a novel crosstalk with NF-κB signaling. To explore the epigenetic regulation of MSC differentiation towards osteoblasts, we discovered two novel histone demethylases KDM4B and KDM6B, which promoted osteogenesis and inhibited adipogenesis of human MSCs. Mechanistically, KDM4B and KDM6B epigenetically activated different osteogenic transcription factors by removing gene silencing marks H3K9me3 and H3K27me3 respectively. Furthermore, H3K27me3- and H3K9me3- positive MSCs in osteoporotic and aged mouse bone marrow become elevated, along with a reduction in KDM4B and KDM6B. These findings supported that these histone demethylases play a critical role in MSC cell fate decisions, and may become potential therapeutic targets for treatment of osteoporosis.