Pseudoshikonin I enhances osteoblast differentiation by stimulating Runx2 and Osterix.

Pseudoshikonin I (PSI), a novel biomaterial isolated from Lithospermi radix, has been recognized as an herbal medicine for the treatment of infectious and inflammatory diseases. Bone remodeling maintains a balance through bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Bone formation is generally attributed to osteoblasts. However, the effects of PSI on the bone are not well known. In this study, we found that the ethanol extracts of PSI induced osteoblast differentiation by increasing the expression of bone morphogenic protein 4 (BMP 4). PSI positively regulates the transcriptional expression and osteogenic activity of osteoblast-specific transcription factors such as Runx2 and Osterix. To identify the signaling pathways that mediate PSI-induced osteoblastogenesis, we examined the effects of serine-threonine kinase inhibitors that are known regulators of Osterix and Runx2. PSI-induced upregulation of Osterix and Runx2 was suppressed by treatment with AKT and PKA inhibitors. These results suggest that PSI enhances osteoblast differentiation by stimulating Osterix and Runx2 via the AKT and PKA signaling pathways. Thus, the activation of Runx2 and Osterix is modulated by PSI, thereby demonstrating its potential as a treatment target for bone disease.

Design and synthesis of novel androgen receptor antagonists via molecular modeling.

Several androgen receptor (AR) antagonists are clinically prescribed to treat prostate cancer. Unfortunately, many patients become resistant to the existing AR antagonists. To overcome this, a novel AR antagonist candidate called DIMN was discovered by our research group in 2013. In order to develop compounds with improved potency, we designed novel DIMN derivatives based on a docking study and substituted carbons with heteroatom moieties. Encouraging in vitro results for compounds 1b, 1c, 1e, 3c, and 4c proved that the new design was successful. Among the newly synthesized compounds, 1e exhibited the strongest inhibitory effect on LNCaP cell growth (IC50=0.35µM) and also acted as a competitive AR antagonist with selectivity over the estrogen receptor (ER) and the glucocorticoid receptor (GR). A docking study of compound 1e fully supported these biological results. Compound 1e is considered to be a novel, potent and AR-specific antagonist for treating prostate cancer. Thus, our study successfully applied molecular modeling and bioisosteric replacement for hit optimization. The methods here provide a guide for future development of drug candidates through structure-based drug discovery and chemical modifications.

Protein kinase A regulates the osteogenic activity of Osterix.

Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

Protein kinase a phosphorylates Dlx3 and regulates the function of Dlx3 during osteoblast differentiation.

Protein kinase A (PKA), a serine/threonine kinase, regulates bone formation, and enhances Bone morphogenetic protein (BMP)-induced osteoblast differentiation. However, the mechanisms of how PKA controls the cellular response to BMP are not well known. We investigated the effects of modulating PKA activity during BMP2-induced osteoblast differentiation, and found that PKA regulates the function of Dlx3. Dlx3 plays crucial roles in osteoblast differentiation and it is expressed in most skeletal elements during development. We found that PKA activation increases BMP2-induced expression of Dlx3 protein, and enhances the protein stability, DNA binding, and transcriptional activity of Dlx3. In addition, PKA activation induces the phosphorylation of Dlx3 at consensus PKA phosphorylation target site(s). Lastly, substitution of serine 10 in Dlx3 to alanine significantly reduces, if not completely abolishes, the phosphorylation of Dlx3 and the regulation of Dlx3 function by PKA. These results suggest that Dlx3 is a novel target of PKA, and that PKA mediates BMP signaling during osteoblast differentiation, at least in part, by phosphorylating Dlx3 and modulating the protein stability and function of Dlx3.

Cbl regulates the activity of SIRT2.

SIRT2 is a member of the sirtuin family of NAD(+)-dependent protein deacetylases. It is involved in metabolic homeostasis and has been linked to the progression of age-related diseases. Casitas B-lineage lymphoma (Cbl) proteins regulate signal transduction through many pathways and, consequently, regulate cell function and development. Cbl proteins are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The function of SIRT2 is modulated by post-translational modifications. However, the precise molecular signaling mechanism of SIRT2 through interactions with Cbl proteins has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by the Cbl mammalian family members Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl increased the protein level and stability of SIRT2 and that Cbl-b and c-Cbl interact with SIRT2. They were also found to regulate the deacetylase activity of SIRT2. Further investigation revealed that Cbl-mediated SIRT2 regulation occurred via ubiquitination of SIRT2.

Src regulates the activity of SIRT2.

SIRT2 is a mammalian member of the Sirtuin family of NAD(+)-dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation.

PKC signaling inhibits osteogenic differentiation through the regulation of Msx2 function.

Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, ß, δ, and ζ) interacted with Msx2, and PKCß phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.

Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix.

Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

ERK1/2 regulates SIRT2 deacetylase activity.

SIRT2 is a mammalian member of the Sirtuin family of NAD-dependent protein deacetylases. The function of SIRT2 can be modulated by post-translational modification. However, the precise molecular signaling mechanisms of SIRT2 and extracellular signal-regulated kinase (ERK)1/2 have not been correlated. We investigated the potential regulation of SIRT2 function by ERK1/2. ERK activation by the over-expression of constitutively active MEK increased protein levels and enhanced the stability of SIRT2. In contrast, U0126, an inhibitor of mitogen-activated kinase kinase, suppressed SIRT2 protein level. ERK1/2 interacted with SIRT2 exogenously and endogenously. Deacetylase activity of SIRT2 was up-regulated in an ERK1/2-mediated manner. These results suggest that ERK1/2 regulates SIRT2 by increasing the protein levels, stability and activity of SIRT2.

Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix.

Risedronate increases osteoblastic differentiation and function through connexin43.

Bisphosphonates are potent antiresorptive drugs which have antifracture efficacy by reducing bone turnover rate and increasing bone mineral density. In addition to inhibiting osteoclast function, bisphosphonates have been reported to also promote survival of osteocyte and osteoblast via an anti-apoptotic effect, mediated by opening of hemi-gap junction channels formed by connexin43 (Cx43). In this study, we investigated the effect of risedronate, one amino-bisphosphonate, on osteoblast differentiation and Cx43 expression using the mesenchymal cell line C2C12. Risedronate dose-dependently increased the activity of osterix (OSE)-luciferase containing Runx2 response element with highest activity at 50µM. The activity of osteocalcin (OC)- and bone sialoprotein (BSP)-luciferase reporters, markers of osteoblast differentiation, were also increased by risedronate. When risedronate and BMP2 were used in combination, alkaline phosphatase (ALP) activity increased to a larger extent than when BMP2 was used alone. Risedronate as well as the pro-osteogenic transcription factors, Runx2, Osterix or Dlx5, increased transcriptional activity of the Cx43 promoter in a dose-dependent manner. In the presence of Runx2 or Dlx5, risedronate had an additive effect on Cx43 promoter activity. Accordingly, risedronate increased protein expression of Cx43, Runx2, Osterix, and Dlx5. These results suggest that risedronate promotes osteoblastic differentiation and positively regulates Cx43 gene transcription.

Functional enhancement of exosomes derived from NK cells by IL-15 and IL-21 synergy against hepatocellular carcinoma cells: The cytotoxicity and apoptosis in vitro study.

Exosomes are released by various cells, including natural killer (NK) cells and transport signaling molecules for the intercellular communication. Hepatocellular carcinoma (HCC), also known as primary liver cancer, is often inoperable and difficult to accurate diagnosis. Notably, the prognosis and underlying mechanisms of HCC are not fully understood. Exosomes-derived NK cells (NK-exos) express unique cytotoxic proteins with a killing ability in tumors and can easily penetrate tumor tissues to improve their targeting ability. NK cell functions, inducing cellular cytotoxicity are modulated by cytokines such as interleukin (IL)-15 and IL-21. However, the mechanisms and effects of cytokines-stimulated NK-exos for the treatment of liver cancer, including HCC, are not well known. In this study, we aimed to investigate the synergistic anti-tumor effects of NK-exos stimulated with IL-15 and IL-21 (NK-exosIL-15/21) in Hep3B cells. Our findings revealed that NK-exosIL-15/21 expressed cytotoxic proteins (perforin and granzyme B) and contained typical exosome markers (CD9 and CD63) within the size range of 100-150 nm. Moreover, we demonstrated that NK-exosIL-15/21 induced the enhancement of cytotoxicity and apoptotic activity in Hep3B cells by activating the specific pro-apoptotic proteins (Bax, cleaved caspase 3, cleaved PARP, perforin, and granzyme B) and inhibiting the anti-apoptotic protein (Bcl-2). In summary, our results suggest that NK-exosIL-15/21 regulate strong anti-tumor effects of HCC cells, by increasing the cytotoxicity and apoptosis through the activation of specific cytotoxic molecules.

Akt1 regulates phosphorylation and osteogenic activity of Dlx3.

Distal-less 3 (DLX3) is a highly conserved homeobox containing transcription factor. DLX3 is specifically expressed in osteoblasts and osteocytes of all developing bones. DLX3 is essential for osteoblast differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. Akt can be activated by several osteogenic signaling molecules, but its precise function and downstream targets in bone development are unknown. In this report, we investigated a potential regulation of Dlx3 function by Akt1. We found that Akt1 phosphorylates Dlx3 and Akt1 activation increases protein stability, osteogenic activity and transcriptional activity of Dlx3. Also, BMP2 was shown to increase the protein level of Dlx3 in an Akt1 activity-dependent manner. Conversely, inhibition of Akt1 by the Akt inhibitor decreases the protein levels of Dlx3. These results suggest that Dlx3 is a novel target of Akt1 and the activity of Dlx3 could be modulated by a novel mechanism involving Akt1 during osteoblast differentiation.

Exploring the dynamic nature of divalent metal ions involved in DNA cleavage by CRISPR-Cas12a.

CRISPR-Cas12a has been widely used in genome editing and nucleic acid detection. In both of these applications, Cas12a cleaves target DNA in a divalent metal ion-dependent manner. However, when and how metal ions contribute to the cleavage reaction is unclear. Here, using a single-molecule FRET assay, we reveal that these metal ions are necessary for stabilising cleavage-competent conformations and that they are easily exchangeable, suggesting that they are dynamically coordinated.

Tumor-derived extracellular vesicles for the active targeting and effective treatment of colorectal tumors in vivo.

Colorectal cancer remains one of the main causes of cancer-related deaths worldwide. Although numerous nanomedicine formulations have been developed to tackle the disease, their low selectivity still limits effective therapeutic outcomes. In this study, we isolated extracellular vesicles (EVs) from CT26 colorectal cancer cells and 4T1 murine mammary carcinoma cells, loaded them with the chemotherapeutic agent (doxorubicin, DOX). Then we evaluated the cellular uptake of the extracellular vesicles both in 2D monolayer and 3D tumor spheroid setups using confocal laser scanning microscope and flow cytometry. In vivo tumor homing of the extracellular vesicles was verified on CT26 tumor bearing BALB/c mice using in vivo imaging system. Finally, in vivo therapeutic effects were evaluated and compared using the same animal models treated with five doses of EV formulations. CT26-EV-DOX exhibited excellent biocompatibility, a high drug-loading capacity, controlled drug release behavior, and a high capability for targeting colorectal cancer cells. In particular, we verified that CT26-EV-DOX could preferentially be up taken by their parent cells and could effectively target and penetrate 3D tumor spheroids resembling colorectal tumors in vivo in comparison with their 4T1 derived EV partner. Additionally, treatment of colorectal tumor-bearing BALB/c mice with of CT26-EV-DOX significantly inhibited the growth of the tumors during the treatment course. The developed CT26-EV-DOX nanoparticles may present a novel and effective strategy for the treatment of colorectal cancer.

Delivery of human natural killer cell-derived exosomes for liver cancer therapy: an in vivo study in subcutaneous and orthotopic animal models.

Exosomes are nanosized extracellular vesicles secreted by various cell types, including those of the immune system, such as natural killer (NK) cells. They play a role in intercellular communication by transporting signal molecules between the cells. Recent studies have reported that NK cell-derived exosomes (NK-exo) contain cytotoxic proteins-induced cell death. However, the characteristics and potential functions of NK-exo, especially for the liver cancer are poorly understood. In this study, we investigated the anti-tumor effects of NK-exo in the primary liver cancer, hepatocellular carcinoma (HCC), using the orthotopic and subcutaneous tumor model. We found that NK-exo expressed both typical exosomal markers (e.g. CD63, CD81, and Alix) and cytotoxic proteins (e.g. perforin, granzyme B, FasL, and TRAIL). NK-exo were selectively taken up by HCC cells (e.g. Hep3B, HepG2, and Huh 7). Interestingly, Hep3B cells induced the highest cytotoxicity compared with HepG2 and Huh7 cells, and substantially enhanced the apoptosis by NK-exo. Furthermore, we demonstrated that NK-exo inhibited the phosphorylation of serine/threonine protein kinases (e.g. AKT and ERK1/2), and enhanced the activation of specific apoptosis markers (e.g. caspase-3, -7, -8, -9, and PARP) in Hep3B cells. NK-exo also exhibit the active targeting ability and potent therapeutic effects in both orthotopic and subcutaneous HCC mouse models. Overall, these results suggest that NK-exo indicate strong anti-tumor effects in HCC, which are mediated by novel regulatory mechanisms involved in serine/threonine kinase pathway-associated cell proliferation and caspase activation pathway-associated apoptosis.

Osterix is regulated by Erk1/2 during osteoblast differentiation.

Osterix (Osx) is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation in bone homeostasis. The mitogen-activated protein (MAP) kinases are a group of evolutionarily conserved proline-directed protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. Erk1/2 plays essential roles in osteoblast differentiation and in supporting osteoclastogenesis, but the precise molecular signaling mechanisms between Osterix and Erk1/2 are not known. We therefore focused on the relationship between Osterix and Erk1/2 during osteoblast differentiation because BMP signaling induces Erk activation in osteoblasts. We investigated the role of the MAPK pathway in regulating protein levels and transcriptional functions of Osterix. We found that Erk activation by overexpression of constitutively active MEK increased the mRNA and protein levels of Osterix and enhanced the transcriptional activity of Osterix, whereas U0126, an inhibitor of MEK, suppressed the protein levels of Osterix and the transcriptional activity. Also, overexpression of constitutively active MEK stabilized Osterix protein. These results suggest that Erk1/2 regulates a major transcription factor, Osterix, during osteoblast differentiation by increasing its protein stability and transcriptional activity.

Akt phosphorylates and regulates the function of Dlx5.

Akt, a phosphoinositide-dependent serine/threonine protein kinase, acts as a key regulator in bone formation. Akt can be activated by several osteogenic signaling molecules, but its precise function and downstream targets in bone development are unknown. Dlx5 transcription factor plays important roles during bone development and osteoblast differentiation. Its expression is regulated by several osteogenic signals. In addition, Dlx5 function is also regulated through post-translational modification by several kinases. In this report, we have investigated a potential regulation of Dlx5 function by Akt. Our results indicate that Akt interacts with and phosphorylates Dlx5. In addition, we provide evidences that Akt kinase activity is important for Akt to enhance the protein stability and transcriptional activity of Dlx5. These results suggest that Dlx5 is a novel target of Akt and that the activity of Dlx5 could be modulated by a novel mechanism involving Akt during osteoblast differentiation.

Xanthohumol from the hop plant stimulates osteoblast differentiation by RUNX2 activation.

Xanthohumol (XN), the principal prenylated flavonoid from the hop plant, an additive that contributes bitterness and flavor to beer, is known to be a potent phytoestrogen. Although XN has been identified as a chemopreventive agent and as an anti-infective agent, its effects on bone are unknown. In the present study, the effects of XN on osteoblast differentiation and function were determined by analyzing the activity of alkaline phosphatase (ALP), an osteoblast marker, and the regulation of RUNX2, a master gene of osteoblast differentiation, in a mesenchymal stem cell line. XN upregulated ALP activity and the expression of osteogenic marker genes. Additionally, XN increased the expression and transcriptional activity of RUNX2. To determine which signaling pathways are involved in the osteogenic effects of XN, we tested the effect of inhibitors of kinases known to regulate RUNX2. Enhancement of the transcriptional activity and expression of RUNX2 were inhibited by treatment with a p38 and an ERK inhibitor. These findings suggest that XN stimulates osteoblast differentiation by activation of RUNX2 via mechanisms related to the p38 MAPK and ERK signaling pathway. Regulation of RUNX2 activation by XN may be an important therapeutic target for osteoporosis.

Akt phosphorylates and regulates the osteogenic activity of Osterix.

Osterix (Osx), a zinc-finger transcription factor is required for osteoblast differentiation and new bone formation during embryonic development. Akt is a member of the serine/threonine-specific protein kinase and plays important roles in osteoblast differentiation. The function of Osterix can be also modulated by post-translational modification. But, the precise molecular signaling mechanisms between Osterix and Akt are not known. In this study, we investigated the potential regulation of Osterix function by Akt in osteoblast differentiation. We found that Akt phosphorylates Osterix and that Akt activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. We also found that BMP-2 increases the protein level of Osterix in an Akt activity-dependent manner. These results suggest that Akt activity enhances the osteogenic function of Osterix, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Osterix, at least in part, through Akt.