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Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.

Broussochalcones A (BCA, 1) and B (BCB, 2) are major bioactive constituents isolated from Broussonetia papyrifera, a polyphenol-rich plant belonging to the family Moraceae. Due to their low yields from natural sources, BCA (1) and BCB (2) were prepared synthetically by employing Claisen-Schmidt condensation, and these were used as substrates for microbial transformation to obtain novel derivatives. Microbial transformation of BCA (1) and BCB (2) with the endophytic fungus Aspergillus niger KCCM 60332 yielded 10 previously undescribed chalcones (1a-1e and 2a-2e). Their structures were established based on the spectroscopic methods. The cytotoxicity of BCA (1), BCB (2), and their metabolites (1a-1e and 2a-2e) was determined by human cancer cell lines A375P, A549, HT-29, MCF-7, and HepG2, with 1e shown to be most cytotoxic.

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms, however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.

Diosgenin (DG) isolated from yam roots revealed various bioactivities and applications as drug carrier. In the present study, a conjugate of DG with cytarabine (Ara-C) was used to prepare the self-assembled nanoparticles (NPs) of DG-Ara-C by a nanoprecipitation method. Dynamic light scattering (DLS) as well as transmission electron microscopy (TEM) were employed to analyze the size and the morphology of NPs, respectively. The stability and absorption of DG-Ara-C NPs were measured. Additionally, the cytotoxicity of the NPs was determined via MTT assay. The results indicated that the average particle size of DG-Ara-C NPs was around 190 nm with a narrow size distribution (PDI = 0.1). TEM showed that DG-Ara-C NPs had a spherical morphology. Compared to free DG or Ara-C, the self-assembled DG-Ara-C NPs exhibited a better anti-tumor activity against solid tumor cells as well as leukemia cells. In conclusion, DG possesses dual role in the self-assembled NPs of DG-Ara-C conjugate, being as a promising anticancer drug and drug carrier.

2-Hydroxymelatonin is a metabolite produced when melatonin 2-hydroxylase catalyzes melatonin. Recent studies have reported the important roles of melatonin in bone metabolism. However, the roles of 2-hydroxymelatonin in bone metabolism remains poorly understood. The purpose of this study is to present evidence of the effect of 2-hydroxymelatonin on osteogenic differentiation in C2C12 cells. In this study, we demonstrated the synergistic stimulating effect of 2-hydroxymelatonin and bone morphogenetic protein (BMP)-4 on osteogenic differentiation in vitro, using alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, qPCR, and luciferase reporter assay. The combination of 2-hydroxymelatonin and BMP-4 revealed a synergistic effect on osteogenic differentiation in vitro. This finding provides evidence that optimal concentrations of both 2-hydroxymelatonin and BMP-4 are beneficial for anabolic effects on bone in vitro.

High-fat diet (HFD)-induced obesity is implicated in diabetic nephropathy (DN). EX-527, a selective Sirtuin 1 (SIRT1) inhibitor, has multiple biological functions; however, its protective effect against DN is yet to be properly understood. This study was aimed to explore the protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. After 21 weeks of continually feeding HFD to the rats, the apparent characteristics of progressive DN were observed, which included an increase in kidney weight (~160%), hyperglycemia, oxidative stress, and inflammatory cytokines, and subsequent renal cell damage. However, the administration of EX-527 for 10 weeks significantly reduced the blood glucose concentration and kidney weight (~59%). Furthermore, EX-527 significantly reduced the serum concentration of transforming growth factor-β1 (49%), interleukin (IL)-1β (52%), and IL-6 in the HFD-fed rats. Overall, the antioxidant activities significantly increased, and oxidative damage to lipids or DNA was suppressed. Particularly, EX-527 significantly reduced blood urea nitrogen (81%), serum creatinine (71%), microalbumin (43%), and urinary excretion of protein-based biomarkers. Histopathological examination revealed expansion of the extracellular mesangial matrix and suppression of glomerulosclerosis following EX-527 administration. EX-527 downregulated the expression of α-SMA (~64%), TGF-β (25%), vimentin, α-tubulin, fibronectin, and collagen-1 in the kidneys of the HFD-fed rats. Additionally, EX-527 substantially reduced claudin-1 and SIRT1 expression, but increased the expression of SIRT3 in the kidneys of the HFD-fed rats. EX-527 also inhibited the growth factor receptors, including EGFR, PDGFR-β, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

Hepatic fibrosis is characterized by persistent deposition of extracellular matrix proteins and occurs in chronic liver diseases. The aim of the present study is to investigate whether estrogen deficiency (ED) potentiates hepatic fibrosis in a thioacetamide (TAA)-treated rat model. Fibrosis was induced via intraperitoneal injection (i.p.) of TAA (150 mg/kg/day) for four weeks in ovariectomized (OVX) female, sham-operated female, or male rats. In TAA-treated OVX rats, the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and &gamma, glutamyl transferase (GGT) were significantly increased compared to those in TAA-treated sham-operated OVX rats or TAA-treated male rats. Furthermore, &alpha, smooth muscle actin (&alpha, SMA) expression was significantly increased compared to that in TAA-treated sham-operated rats. This was accompanied by the appearance of fibrosis biomarkers including vimentin, collagen-I, and hydroxyproline, in the liver of TAA-treated OVX rats. In addition, ED markedly reduced total glutathione (GSH) levels, as well as catalase (CAT) and superoxide dismutase (SOD) activity in TAA-treated OVX rats. In contrast, hepatic malondialdehyde (MDA) levels were elevated in TAA-treated OVX rats. Apoptosis significantly increased in TAA-treated OVX rats, as reflected by elevated p53, Bcl-2, and cleaved caspase 3 levels. Significant increases in interleukin-6 (IL-6) and tumor necrosis factor-&alpha, (TNF-&alpha, ) concentrations were exhibited in TAA-treated OVX rats, and this further aggravated fibrosis through the transforming growth factor-&beta, (TGF-&beta, )/Smad pathway. Our data suggest that ED potentiates TAA-induced oxidative damage in the liver, suggesting that ED may enhance the severity of hepatic fibrosis in menopausal women.

Osteoporosis is a degenerative metabolic disease caused by an imbalance between osteogenesis and osteoclastogenesis. Increased levels of proinflammatory cytokines combined with decreased estrogen levels, which are commonly seen in postmenopausal women, can lead to overactivation of osteoclasts. Therefore, targeting osteoclast maturation may represent a novel strategy for both treating and preventing osteoporosis. Auranofin is a gold-based compound first approved in 1985 for the treatment of rheumatic diseases. Here, we examined whether auranofin suppresses osteoclast differentiationin vitroandin vivo. Auranofin was shown to suppress receptor activator of NF-κB ligand- (RANKL-) induced osteoclastogenesis in mouse bone marrow macrophages (BMMs) and Raw264.7 macrophages. Cotreatment of macrophages with auranofin blocked the RANKL-induced inhibitors ofκB kinase (IKK) phosphorylation, resulting in inhibition of nuclear translocation of p65. The pan-caspase inhibitor nivocasan potently reduced not only inflammasome-mediated interleukin-1β(IL-1β) secretion but also osteoclast differentiation in BMMs. Auranofin suppressed inflammasome activation, as evidenced by decreased production of cleaved IL-1βin both bone marrow-derived macrophages (BMDMs) and J774.A1 cells. Loss of both bone mass in ovariectomized mice was significantly recovered by oral administration of auranofin. Taken together, these data strongly support the use of auranofin for the prevention of osteoclast-related osteoporosis.

Diosgenin (DG), a steroidal saponin, is mainly found in yam tubers. DG and its derivatives displayed significant pharmacological activities against inflammatory, hyperlipidemia, and various cancers. DG was selected to modify the cancer chemotherapeutic agent cytarabine (Ara-C) due to its anti-tumor activities as well as lipophilicity. After characterization, the biomembrane affinity and the kinetic thermal processes of the obtained DG-Ara-C conjugate were evaluated by differential scanning calorimetry (DSC). Thin hydration method with sonication was applied to prepare the DG-Ara-C liposomes without cholesterol since the DG moiety has the similar basic structure with cholesterol with more advantages. Dynamic Light Scattering (DLS) analysis and cytotoxic analysis were employed to characterize the DG-Ara-C liposomes and investigate their biological activities, respectively. The results indicated that DG changed the biomembrane affinity of Ara-C and successfully replaced the cholesterol during the liposome preparation. The DG-Ara-C liposomes have an average particle size of around 116 nm with a narrow size distribution and revealed better anti-cancer activity against leukemia cells and solid tumor cells than that of free DG or Ara-C. Therefore, it can be concluded that DG displayed the potential application as an anti-cancer drug carrier to improve the bio-activities, since DG counted for a critical component in modulating the biomembrane affinity, preparation of liposome, and release of hydrophilic Ara-C from lipid vesicles.

Platycodon grandiflorum root-derived saponins (Changkil saponins, CKS) are reported to have many pharmacological activities. In our latest research, CKS was proven to have a significant osteogenic effect. However, the detail molecular mechanism of CKS on osteoclastic differentiation has not been fully investigated. Administration of CKS considerably reduced OVX-induced bone loss, and ameliorated the reduction in plasma levels of alkaline phosphatase, calcium, and phosphorus observed in OVX mice. CKS also repressed the deterioration of bone trabecular microarchitecture. Interestingly, platycodin D, the most abundant and major pharmacological constituent of triterpenoid CKS, inhibited receptor activator of NF-κB ligand (RANKL)-induced activation of NF-κB, and ERK and p38 MAPK, ultimately repressing osteoclast differentiation. OVX-induced bone turnover was attenuated by CKS, possibly via repression of osteoclast differentiation by platycodin D, the active component of CKS. Platycodin D can be regarded as an antiosteoporotic candidate for treatment of osteoporosis diseases. J. Cell. Biochem. 118: 860-868, 2017. © 2016 Wiley Periodicals, Inc.

Pin1 is a peptidylprolyl cis/trans isomerase and it has a unique enzymatic activity of catalyzing isomerization of the peptide bond between phospho-serine/threonine and proline. Through the conformational change of its substrates, Pin1 regulates diverse biological processes including adipogenesis. In mouse embryonic fibroblasts and 3T3-L1 preadipocytes, overexpression of Pin1 enhances adipocyte differentiation whereas inhibition of Pin1 activity suppresses it. However, the precise functions of Pin1 during adipogenesis are not clear. In the present study, we investigated the potential targets of Pin1 during adipogenesis. We found that Pin1 interacts directly with and regulates the transcriptional activity of PPARγ, a key regulator of adipogenesis. In addition, ERK activity and Ser273 of PPARγ, a potential ERK phosphorylation target site, are important for the regulation of PPARγ function by Pin1 in 3T3-L1 cells. Taken together our results suggest a novel regulatory mechanism of Pin1 during adipogenesis, in which Pin1 enhances adipocyte differentiation by regulating the function of PPARγ.

Osterix is an essential transcription factor for osteogenesis and is expressed in osteoblasts. Although Osterix has been shown to be induced by bone morphogenetic protein 4, the molecular mechanism underlying Osterix function during osteoblast differentiation remains unclear. Connexin43 (Cx43) is the most abundant gap junction protein in bone cells and plays a critical role in osteoblast differentiation. However, little is known about the functional interactions between Osterix and the Cx43 promoter. In the present study, we investigated the relationship between Osterix and Cx43 in HEK293 and C2C12 cells. Cx43 expression was significantly repressed by the addition of shRNA against Osterix, whereas over- expression of Osterix resulted in enhanced Cx43 expression. Furthermore, Osterix directly occupied the promoter region of Cx43 and subsequently increased Cx43 promoter activity in a dose-dependent manner. In addition, phosphorylation of the Ser76 and Ser80 residues in Osterix were found to be critical for its activity on the Cx43 promoter. Our results suggest that Osterix plays an important role in increasing bone morphogenetic protein 4-induced Cx43 activity. ⓒ 2016 Elsevier Inc. All rights reserved.

Previous studies have suggested that platycodin D is implicated in bone biology and ameliorates osteoporosis development. Platycodin D repressed the osteoclast activity and enhanced bone mineral density in the mouse model. However, the effects of platycodin D on osteoblast differentiation have not been elucidated yet. In C3H10T1/2 cells, platycodin D upregulated osteogenic markers including alkaline phosphatase (ALP), bone sialoprotein, and collagen type 1 alpha 1, and transcription factors, such as Runx2 and osterix, subsequently enhancing the bone mineralization. In a molecular mechanism study, platycodin D induced β-catenin nuclear accumulation by upregulating GSK3β phosphorylation. Furthermore, platycodin D upregulated the ALP activity and enhanced the mineralization process in osteoblast cells via the sirtuin 1/β-catenin pathways. Taken together, these results suggested that platycodin D could be an effective therapeutic compound against osteoporosis because of its regulatory effects during the osteoblast differentiation.

Berberine has been implicated to be involved in maintaining bone health due to its anti-oxidative and osteogenic properties. However, low potency and low bioavailability limit the clinical development of the drug. To overcome these obstacles, we previously synthesized a compound, Q8, which is a structural homolog of berberine. The present study examined the pharmacological functions of Q8 to evaluate its potential use in bone regeneration with respect to osteoblast differentiation. Here, we report that Q8 enhanced BMP4-induced alkaline phosphatase (ALP) activity and transcription from the ALP promoter. In addition, Q8 suppressed the expression and activity of PPARγ (a known negative regulator of osteogenesis due to its stimulatory effects on adipogenesis and its role as an adipogenic transcription factor), which in turn increases β-catenin expression in the nucleus, and ultimately promotes osteoblast differentiation. Meanwhile, Q8 reversed the inhibitory effects of the PPARγ agonist, rosiglitazone, on osteoblast differentiation. This study demonstrated that Q8 promotes osteoblast differentiation via inhibition of PPARγ and the enhancement of osteoblast function by Q8 may contribute to the prevention for osteoporosis.

The objective of this study was to elucidate the effect of hepatic damage on cisplatin (CDDP)-induced acute kidney injury (AKI). Thioacetamide (TAA, 150 mg/kg), a hepatotoxicant, was intraperitoneally (i.p.) injected to male Sprague-Dawley rats for 3 d prior to CDDP (5 mg/kg, i.p.) injection. All animals were sacrificed 5 d after CDDP treatment, and urine or blood was obtained to measure various parameters. No significant changes in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were observed after CDDP treatment. However, pretreatment with TAA significantly elevated ALT and AST activity. Serum blood urea nitrogen and creatinine levels significantly increased in CDDP-treated group compared to control. In addition, urinary excretion of novel protein-based biomarkers such as neutrophil gelatinase-associated lipocalin, vascular endothelial growth factor, kidney injury molecule-1, and tissue inhibitor of metalloproteinase-1 rose markedly in the CDDP-treated group. In particular, pretreatment with TAA markedly elevated CDDP-induced urinary excretion of protein-based nephrotoxic biomarkers compared with CDDP alone. Hematoxylin and eosin staining demonstrated that pretreatment with TAA following CDDP injection led to more severe tubular damage and apoptosis in rats compared with CDDP alone. Antioxidant status was significantly reduced in kidneys following pretreatment with TAA prior to CDDP. These findings indicate that liver injury enhanced the vulnerability of kidney to CDDP-induced AKI and this phenomenon may be associated with severe apoptotic damage.

Benign prostatic hyperplasia (BPH) is one of the leading causes of male reproductive disorders. Therapeutic agents currently in use have severe side effects; therefore, alternative drugs that exhibit improved therapeutic activity without side effects are required. The present study investigated the protective effect of GV1001 against testosterone‑induced BPH in rats. BPH in castrated rats was established via daily subcutaneous (s.c.) injections of testosterone propionate (TP, 3 mg/kg) dissolved in corn oil for 4 weeks. GV1001 (0.01, 0.1 and 1 mg/kg, s.c.) was administered 3 times per week for 4 weeks, together with TP (3 mg/kg) injection. The rats were sacrificed on the last day of treatment, and their prostates were excised and weighed for biochemical and histological studies. Serum levels of testosterone and dihydrotestosterone (DHT) were also measured. In rats with TP‑induced BPH, a significant increase in prostate weight (PW) and prostatic index (PI), accompanied by a decrease in antioxidant enzyme activity, was observed. Histological studies revealed clearly enlarged glandular cavities in rats with BPH. GV1001 (0.01 and 0.1 mg/kg) treatment significantly decreased PW and PI in rats with TP‑induced BPH. In addition, GV1001 demonstrated a potent inhibitory effect on 5α‑reductase in prostate. The present data suggest that the protective role of GV1001 against testosterone‑induced BPH is closely associated with its antioxidant potential. Additional studies are required to identify the mechanisms by which GV1001 protects against BPH to determine its clinical application.

Osterix, a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. The c-Src of tyrosine kinase is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. Src activity is tightly regulated and its dysregulation leads to constitutive activation and cellular transformation. The function of Osterix can be also modulated by post-translational modification. But the precise molecular signaling mechanisms between Osterix and c-Src are not known. In this study we investigated the potential regulation of Osterix function by c-Src in osteoblast differentiation. We found that c-Src activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. The siRNA-mediated knockdown of c-Src decreased the protein levels and transcriptional activity of Osterix. Conversely, Src specific inhibitor, SU6656, decreased the protein levels and transcriptional activity of Osterix. The c-Src interacts with and phosphorylates Osterix. These results suggest that c-Src signaling modulates osteoblast differentiation at least in part through Osterix.

Osterix is an essential transcription factor for osteoblast differentiation and bone formation. The mechanism of regulation of Osterix by post-translational modification remains unknown. Peptidyl-prolyl isomerase 1 (Pin1) catalyzes the isomerization of pSer/Thr-Pro bonds and induces a conformational change in its substrates, subsequently regulating diverse cellular processes. In this study, we demonstrated that Pin1 interacts with Osterix and influences its protein stability and transcriptional activity. This regulation is likely due to the suppression of poly-ubiquitination-mediated proteasomal degradation of Osterix. Collectively, our data demonstrate that Pin1 is a novel regulator of Osterix and may play an essential role in the regulation of osteogenic differentiation.

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. This article is protected by copyright. All rights reserved

Transcription factor YY1 plays important roles in cell proliferation and differentiation. For example, YY1 represses the expression of muscle-specific genes and the degradation of YY1 is required for myocyte differentiation. The activity of YY1 can be regulated by various post-translational modifications; however, little is known about the regulatory mechanisms for YY1 degradation. In this report, we attempted to identify potential E3 ubiquitin ligases for YY1, and found that Smurf2 E3 ubiquitin ligase can negatively regulate YY1 protein level, but not mRNA level. Smurf2 interacted with YY1, induced the poly-ubiquitination of YY1 and shortened the half-life of YY1 protein. Conversely, an E3 ubiquitin ligase-defective mutant form of Smurf2 or knockdown of Smurf2 increased YY1 protein level. PPxY motif is a typical target recognition site for Smurf2, and the PPxY motif in YY1 was important for Smurf2 interaction and Smurf2-induced degradation of YY1 protein. In addition, Smurf2 reduced the YY1-mediated activation of a YY1-responsive reporter whereas Smurf2 knockdown increased it. Finally, Smurf2 relieved the suppression of p53 activity by YY1. Taken together, our results suggest a novel regulatory mechanism for YY1 function by Smurf2 in which the protein stability and transcriptional activity of YY1 are regulated by Smurf2 through the ubiquitin-proteasome-mediated degradation of YY1.

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. J. Cell. Biochem. 115: 1808–1815, 2014. © 2014 Wiley Periodicals, Inc.

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.

Runx2 is a major transcription factor that induces osteoblast differentiation by bone morphogenetic proteins (BMPs). Conversely, YY1 is a transcription factor that inhibits BMP2-induced cell differentiation. Until now, there has been no understanding of how osteoblast differentiation by Runx2 and YY1 is regulated. In this study we focused on the relationship between Runx2 and YY1. We confirmed that alkaline phosphatase staining is repressed by YY1. Runx2 interacted with YY1 through Runt and the C-terminus domain of Runx2. YY1 markedly repressed the Runx2-mediated enhancement of transcriptional activity on the osteocalcin and alkaline phosphatase promoters. Knockdown of YY1 enhanced BMP2- and Runx2-induced osteoblast differentiation. YY1 decreased Runx2 DNA binding affinity. The results indicate that YY1 represses osteoblast differentiation by an interaction with Runx2 and inhibits the transcriptional activity of Runx2.

Use of the chemotherapeutic agent cisplatin (CDDP) in cancer patients is limited by the occurrence of acute kidney injury (AKI), however, no protective therapy is available. We aimed to investigate the renoprotective effects of Dendropanax morbifera water extract (DM) on CDDP-induced AKI. Male Sprague-Dawley rats (six animals/group) received: Vehicle (control), CDDP (6 mg/kg, intraperitoneally (i.p.), DM (25 mg/kg, oral), or DM + CDDP injection. CDDP treatment significantly increased blood urea nitrogen (BUN), serum creatinine (sCr), and pro-inflammatory cytokines (IL-6 and TNF-&alpha, ), and severely damaged the kidney architecture. Urinary excretion of protein-based AKI biomarkers also increased in the CDDP-treated group. In contrast, DM ameliorated CDDP-induced AKI biomarkers. It markedly protected against CDDP-induced oxidative stress by increasing the activity of endogenous antioxidants and reducing the levels of pro-inflammatory cytokines (IL-6 and TNF-&alpha, ). The protective effect of DM in the proximal tubules was evident upon histopathological examination. In a tumor xenograft model, administration of DM enhanced the chemotherapeutic activity of CDDP and exhibited renoprotective effects against CDDP-induced nephrotoxicity without altering chemotherapeutic efficacy. Our data demonstrate that DM may be an adjuvant therapy with CDDP in solid tumor patients to preserve renal function.

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.

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.

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.

The Q8 compound is a unique derivative of berberine. The present study investigated the functional role of Q8 to evaluate its potential for use in bone regeneration, especially in osteoblast differentiation. The safe concentration of Q8 increased BMP4-induced alkaline phosphatase (ALP) activity, and induced RNA expression of ALP, bone sialoprotein (BSP), and osteocalcin (OC). The activities of ALP-, BSP- and OC-luciferase reporters were also increased by Q8. During osteoblast differentiation, Q8 stabilized the Runx2 and Osterix protein abundance by blocking the ubiquitin-proteasome pathway, which in turn promoted Runx2 and Osterix induced transcriptional activity and subsequently increased the osteoblast differentiation. Meanwhile, depletion of Runx2 and Osterix markedly abolished the bone anabolic effect of Q8 on osteoblast differentiation. To evaluate the signal transduction pathway involved in the Q8-mediated regulation of Runx2 and Osterix, we examined the reporter assay using various kinase inhibitors. Treatment with a protein kinase A (PKA) inhibitor, H89 inhibited the Q8-mediated regulation of Runx2 and Osterix. Based on these findings, this study demonstrates that Q8 promotes the osteoblast differentiation by stabilization of Runx2/Osterix through the increased activation of PKA signaling. The enhancement of osteoblast function by Q8 may contribute to the prevention for osteoporosis.

Conformational change in helix 12 can alter ligand-induced PPARγ activity; based on this reason, isoquinolinoquinazolinones, structural homologs of berberine, were designed and synthesized as PPARγ antagonists. Computational docking and mutational study indicated that isoquinolinoquinazolinones form hydrogen bonds with the Cys285 and Arg288 residues of PPARγ. Furthermore, SPR results demonstrated strong binding affinity of isoquinolinoquinazolinones towards PPARγ. Additionally, biological assays showed that this new series of PPARγ antagonists more strongly inhibit adipocyte differentiation and PPARγ2-induced transcriptional activity than GW9662.

Chalcones, the biosynthetic precursors of flavonoids and isoflavonoids abundant in edible plants, possess a number of pharmacological properties, and there is growing evidence that chalcone derivatives inhibit TNF-α mediated insulin resistance. The aim of the present study was to define the effects of 4-methoxychalcone (4-MC) on adipocyte differentiation and to determine the underlying molecular mechanism. We investigated the effects of 4-MC on adipocyte differentiation and lipid accumulation, and expression of adipogenic genes in 3T3-L1 cells. Additionally, treatment with 4-MC significantly increased the PPARγ-induced transcriptional activity and 4-MC also enhanced the DNA binding affinity of PPARγ to the proliferator-activated receptor response elements (PPRE) at target promoters. Next, we tested the effect of 4-MC on the inhibition induced by TNF-α on adipocyte differentiation. Treatment with 4-MC enhanced the lipid accumulation and strongly up-regulated the expression of adipogenic markers, including PPARγ, aP2, FAS, and adiponectin during adipocyte differentiation. Finally, 4-MC attenuated the inhibitory effect of TNF-α on adipocyte differentiation and adiponectin expression and subsequently regulated the expression and secretion of various adipokines that are involved in insulin sensitivity. This study clearly demonstrates that 4-MC enhanced adipocyte differentiation, in part, by its potent effects on PPARγ activation and by its reverse effect on TNF-α.

Osterix is a novel bone-related transcription factor involved in osteoblast differentiation, and bone maturation. Because a reciprocal relationship exists between adipocyte and osteoblast differentiation of bone marrow derived mesenchymal stem cells, we hypothesized that Osterix might have a role in adipogenesis. Ablation of Osterix enhanced adipogenesis in 3T3-L1 cells, whereas overexpression suppressed this process and inhibited the expression of adipogenic markers including CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Further studies indicated that Osterix significantly decreased PPARγ-induced transcriptional activity. Using co-immunoprecipitation and GST-pull down analysis, we found that Osterix directly interacts with PPARγ. The ligand-binding domain (LBD) of PPARγ was responsible for this interaction, which was followed by repression of PPARγ-induced transcriptional activity, even in the presence of rosiglitazone. Taken together, we identified the Osterix has an important regulatory role on PPARγ activity, which contributed to the mechanism of adipogenesis.

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.

Many clinical trials have demonstrated the beneficial effects of garlic (Allium sativum) on general cardiovascular health. Aged garlic extract (AGE) is known to display diverse biological activities such as in antioxidant, anti-inflammatory and anticancer activities. However, few studies have been directed on the effect of AGE on cardiovascular function. In this study, we aimed to investigate the effect of AGE and its components on platelet activation, a key contributor in thrombotic diseases. In freshly isolated rat platelets, AGE and its components have shown inhibitory activities on thrombin-induced platelet aggregation. These in vitro results were further confirmed in an in vivo platelet aggregation measurement where tail vein injection of garlic oil and S-Allylmercapto-cysteine (SAMC) significantly reduced thrombin and ADP-induced platelet aggregation. Potential active components for antiplatelet effects of AGE were identified to be SAMC and diallyl sulphide through agonist-induced platelet aggregation assay. These results indicate that aged garlic extract can be a novel dietary supplement for the prevention of cardiovascular risks and the improvement of blood circulation.

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.

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.

JAK2 is an important target in multiple processes associated with tumor growth. In this study, virtual screening was employed for hit compound identification with chemical libraries using SurflexDock. Subsequently, hit optimization for potent and selective candidate JAK2 inhibitors was performed through synthesis of diverse C-1 substituted quinazoline derivatives. A novel compound 5p , (6,7-dimethoxyquinazolin-4-yl)naphthalen-1-ylamine, was thus obtained. JAK2 inhibitory activity of 5p was 43% at 20 μM and this was comparable to AG490, a representative JAK2 inhibitor. Moreover, 5p showed a positive correlation between JAK2 inhibition and cytotoxicity; 5p treatment in HT-29 cells strongly inhibited JAK2 activation and subsequent STAT3 phosphorylation, reduced anti-apoptotic protein levels, and finally induced apoptosis. This suggests that compound 5p is a candidate inhibitor of JAK2 and its downstream STAT3 signaling pathway for antitumor therapy. In the docking model, the quinazoline template of 5k , the lead compound, occupied a hydrophobic region such as Leu856, Leu855, Ala880, Leu932 and Gly935, and the highly conserved hydrogen bond was created by 6-OMe of the ring template, which binds to the NH of Arg980. Moreover, hydrophobic interactions were identified between morpholine moiety and the hydrophobic region formed by Leu855, Ala880, Tyr931, Val911 and Met929. Also, compound 5k more strongly inhibited JAK2 phosphorylation in mouse embryonic stem cells than AG490. Our study shows the successful application of virtual screening for lead discovery and we propose that the novel compound 5p can be an effective JAK2 inhibitor candidate for further antitumor agent research.

Interleukin (IL)-4 plays a key role in T helper 2 (Th2) cell differentiation favoring humoral immune response. Regulation of IL-4 gene expression, therefore, is critically important for Th2 dependent responses and Th2 dominant disorders. In T cells, IL-4 gene expression is regulated positively or negatively by a combination of several transcription factors. Recently, enhanced IL-4 production was reported in Runx3 knockout mice; this implies negative regulation of IL-4 by Runx3. Runx proteins are transcription factors that have a Runt domain and have essential functions in development. In this study, the molecular mechanism that downregulates IL-4 expression was investigated. Runx3 inhibited IL-4 production in EL-4 T cells stimulated with PMA/ionomycin. Runx3-mediated IL-4 inhibition was NFAT-dependent, and Runx3 was physically associated with NFAT. Therefore, our results suggest that the interaction between NFAT and Runx3 is a mechanism that causes the negative regulation of IL-4, along with previously reported repression by T-bet.

Xanthohumol (XN) is a major chalcone found in hop, which is used to add bitterness and flavor to beer. In this study, we investigated the effects of XN on the production of interlukin-2 (IL-2), a potent T cell growth factor. Treatment with XN significantly increased IL-2 production in mouse EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) in a dose-dependent manner. To further characterize its regulatory mechanism of XN on increased IL-2 production, the effects of XN on IL-2 promoter activity and the activity of several transcription factors modulating IL-2 expression were analyzed. XN enhanced activity of the IL-2 promoter, which contains distal and proximal regulatory elements in PMA/Io-activated EL-4 T cells. Furthermore, the activity of NF-AT and AP-1 was enhanced but NF-kappaB activity was not influenced by XN in PMA/Io-activated EL-4 T cells. These results suggest that XN increased IL-2 production at the transcriptional levels via the up-regulation of NF-AT and AP-1 in PMA/Io-activated EL-4 T cells.

Histone deacetylases (HDACs) are subdivided into three classes—HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.

Sirt2 is a mammalian member of the Sirtuin family of NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 beta and gamma among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 beta/gamma augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 beta/gamma. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 beta/gamma is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression.

Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, beta-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both beta-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.

Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPβ (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPβ from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.

Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNFalpha-induced monocytes to endothelial cells and suppressed the TNFalpha-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNFalpha-induced JAK2-PI3K/Akt-NF-kappaB activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.

The Runt domain transcription factors (RUNXs) play essential roles in normal development and neoplasias. Genetic analyses of animals and humans have revealed the involvement of RUNX1 in hematopoiesis and leukemia, RUNX2 in osteogenesis and cleidocranial dysplasia, and RUNX3 in the development of T-cells and dorsal root ganglion neurons and in the genesis of gastric cancer. Here we report that RUNX3 is a target of the acetyltransferase activity of p300. The p300-dependent acetylation of three lysine residues protects RUNX3 from ubiquitin ligase Smurf-mediated degradation. The extent of the acetylation is up-regulated by the transforming growth factor-beta signaling pathway and down-regulated by histone deacetylase activities. Our findings demonstrate that the level of RUNX3 protein is controlled by the competitive acetylation and deacetylation of the three lysine residues, revealing a new mechanism for the posttranslational regulation of RUNX3 expression.

To investigate the nephrotoxic potential of melamine (MEL) and cyanuric acid (CA) in male Sprague-Dawley rats, 7-d repeated-dose studies were performed. The experimental groups of MEL100 and CA100 were orally administered with MEL and CA at 100 mg/kg/d for 7 d, respectively. In groups dosed with MEL-CA mixtures, melamine and cyanuric acid (1:1) were simultaneously administered at 4, 20, or 100 mg/kg/d for 7 d (i.e., MEL-CA4, MEL-CA20, or MEL-CA100, respectively). Body weights were not markedly affected in MEL100, CA100, and MEL-CA4 groups, but significantly reduced in MEL-CA 20 and 100 rats. Most parameters determined in sera and tissues were not markedly altered in MEL100, CA100, and MEL-CA4-treated rodents. However, BUN, creatinine, total protein, and kidney weights were significantly increased in MEL-CA20- and MEL-CA100-treated animals. Renal histopathologic findings also revealed signs of toxicity, including tubular dilatation, crystal deposition, granulomatous tubulo-interstitial inflammation, and tubular necrosis with regeneration. Data suggested that the combination of MEL and CA might be responsible for observed nephrotoxicity that was not seen following individual exposure to either MEL or CA alone. Subsequently, the concentrations of MEL and CA were determined in serum, urine, and kidney tissues by using liquid chromatography-mass spectrometry. Toxicokinetic studies indicated that MEL or CA alone might be eliminated almost completely within 24 h after dosing showing no accumulation in kidney. However, the combined MEL-CA dose produced marked accumulation of chemicals in blood and kidneys. These results suggested that combined MEL and CA might produce renal toxicity due to significant chemical accumulation in kidney accompanied by low excretion.

E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin-proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin-proteasome pathway.

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.

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.

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.

Ginsenoside-Rg1 (G-Rg1) from the roots of Panax ginseng C. A. Meyer has been shown to bind to the glucocorticoid receptor (GR). To further explore the effect of G-Rg1 binding to GR, a luciferase reporter gene containing two copies of a glucocorticoid response element was constructed and transiently transfected into FTO2B rat hepatoma cells. A dose-dependent induction of the reporter gene was observed in response to G-Rg1, and the inductive effect was blocked by treatment with the antiglucocorticoid RU486. In addition, both G-Rg1- and dexamethasone (Dex)-induced transcription was synergistically enhanced by the treatment of dibutyryl cAMP (Bt2-cAMP). G-Rg1 treatment also led to the down-regulation of intracellular GR content, which was similar to the effect of Dex. By showing that G-Rg1 down-regulates GR and induces GR-mediated transcription synergistically with cAMP, we conclude that G-Rg1 is a functional GR ligand in FTO2B cells.