Search

Chronic liver diseases are associated with the development of liver fibrosis. Without treatment, liver fibrosis commonly leads to cirrhosis and HCC. FGF12 is an intracrine factor belonging to the FGF superfamily, but its role in liver homeostasis is largely unknown. This study aimed to investigate the role of FGF12 in the regulation of liver fibrosis.FGF12 was up-regulated in bile duct ligation (BDL)-induced and CCLFGF12 promotes liver fibrosis progression. Therapeutic approaches to inhibit macrophage FGF12 may be used to combat liver fibrosis in the future.

Background Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool. Objectives Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1. Methods Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope. Results Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors. Conclusion In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

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.

Epithelium-specific ETS transcription factor 1 (ESE1) has been implicated in epithelial homeostasis, inflammation, as well as tumorigenesis, and cancer progression. However, numerous studies have reported contradictory roles-as an oncogene or a tumor suppressor of ESE1 in different cancers, and its function in the development and progression of pancreatic ductal adenocarcinoma (PDAC) has remained largely unexplored. Herein, we report that ESE1 was found upregulated in primary PDAC compared to normal pancreatic tissue, but high expression of ESE1 correlated to better relapse-free survival in patients with PDAC. Interestingly, ESE1 was found to exhibit dual roles in regulation of malignant properties of PDAC cells in that its overexpression promoted cell proliferation, whereas its downregulation enhanced epithelial-mesenchymal transition (EMT) phenotype. In the context of TGF-β-induced EMT, ESE1 is markedly downregulated at post-transcriptional level, and reconstituted ESE1 expression partially reversed TGF-β-induced EMT marker expression. Furthermore, we identify AGR2 as a novel transcriptional target of ESE1 that participates in TGF-β-induced EMT in PDAC. Collectively, our findings reveal an ESE1/AGR2 axis that interacts with TGF-β signaling to modulate EMT phenotype in PDAC.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

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.

Hedgehog (Hh) signaling pathway plays an essential role in embryonic development, tissue regeneration, and stem cell renewal. In particular, terminal effectors of the Hh signaling pathway are associated with the regulation of glioma-associated oncogene homolog 1 (GLI1) transcription factors. Overexpression of GLI1 is closely associated with poor prognosis in breast cancer. The Hh-GLI1 signaling pathway is activated and participates in the tumorigenesis and progression of breast cancer, especially in the aggressive subtype of triple-negative breast cancer (TNBC). However, the role of GLI1 in regulating TNBC metabolism remains unclear. This study aimed to explore the functional role of GLI1 in glycolytic metabolism in TNBC. Immunohistochemical analysis of GLI1 expression in a tissue microarray revealed significant correlations between GLI1 expression and advanced tumor stage and grade. GLI1 expression levels were drastically increased in MDA-MB-231 cells compared to those in other cell lines. Inhibition of GLI1 expression using GLI1 small interfering RNA (siRNA) in MDA-MB-231 cells resulted in a significant reduction in cell proliferation and induced cell cycle arrest at the G1 phase. Furthermore, GLI1 downregulation significantly reduced the expression of glycolysis-regulated proteins. GLI1 knockdown resulted in reduced glycolytic rates and extracellular lactate levels. Moreover, metabolic stress after GLI1 knockdown activated the energy sensor, adenosine monophosphate-activated protein kinase, which subsequently resulted in autophagy induction. In conclusion, this study indicates that targeting GLI1 reprograms the tumor glucose metabolism to suppress breast cancer cell growth and proliferation.

Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool.Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope.Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors.In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

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.

Hepatic ischemia-reperfusion injury (IRI) is a major complication of liver surgery and transplantation. IRI leads to hepatic parenchymal cell death, resulting in liver failure, and lacks effective therapeutic approaches. Fibroblast growth factor 10 (FGF10) is a paracrine factor which is well-characterized with respect to its pro-proliferative effects during embryonic liver development and liver regeneration, but its role in hepatic IRI remains unknown. In this study, we investigated the role of FGF10 in liver IRI and identified signaling pathways regulated by FGF10. In a mouse model of warm liver IRI, FGF10 was highly expressed during the reperfusion phase. In vitro experiments demonstrated that FGF10 was primarily secreted by hepatic stellate cells and acted on hepatocytes. The role of FGF10 in liver IRI was further examined using adeno-associated virus-mediated gene silencing and overexpression. Overexpression of FGF10 alleviated liver dysfunction, reduced necrosis and inflammation, and protected hepatocytes from apoptosis in the early acute injury phase of IRI. Furthermore, in the late phase of IRI, FGF10 overexpression also promoted hepatocyte proliferation. Meanwhile, gene silencing of FGF10 had the opposite effect. Further studies revealed that overexpression of FGF10 activated nuclear factor-erythroid 2-related factor 2 (NRF2) and decreased oxidative stress, mainly through activation of the phosphatidylinositol-3-kinase/AKT pathway, and the protective effects of FGF10 overexpression were largely abrogated in NRF2 knockout mice. These results demonstrate the protective effects of FGF10 in liver IRI, and reveal the important role of NRF2 in FGF10-mediated hepatic protection during IRI., Graphical abstract Image 1, Highlights • FGF10 is markedly upregulated in the early phase of liver IRI. • FGF10 overexpression exerts great potential in ameliorating hepatic IRI. • FGF10 knockdown significantly aggravates hepatic IRI. • FGF10 overexpression activates PI3K/AKT-NRF2 signaling and thus ameliorates hepatic IRI. • NRF2 knockout abrogates the protective effects of FGF10 overexpression during liver IRI.

Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation. YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.

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.

Melanoma is among the most aggressive and treatment-resistant human cancers. Aberrant histone H3 methylation at Lys 9 (H3K9) correlates with carcinogenic gene silencing, but the significance of suppressor of variegation 3-9 homolog 1 (SUV39H1), an H3K9-specific methyltransferase, in melanoma initiation and progression remains unclear. Here, we show that SUV39H1-mediated H3K9 trimethylation facilitates retinoblastoma ( RB) 1 promoter CpG island methylation by interacting with DNA methyltransferase 3A and decreasing RB mRNA and protein in melanoma cells. Reduced RB abundance, in turn, impairs E2F1 transcriptional inhibition, leading to increased peptidyl-prolyl cis-trans isomerase never-in-mitosis A (NIMA)-interacting 1 (PIN1) levels, human keratinocyte neoplastic cell transformation, and melanoma tumorigenesis via enhanced rapidly accelerated fibrosarcoma 1(RAF1)-MEK-ERK signaling pathway activation. In a synergistic model with B16-F1 murine melanoma cells, SUV39H1 and PIN1 overexpression increased melanoma growth, which was abrogated by their inhibition in SUV39H1-overexpressing B16-F1 mice. SUV39H1 also positively correlated with PIN1 expression in human melanoma. Our studies establish SUV39H1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.-Kim, G., Kim, J.-Y., Lim, S.-C., Lee, K. Y., Kim, O., Choi, H. S. SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development.

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.

Bone remodeling and homeostasis are largely the result of the coordinated action of osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation. The differentiation of osteoblasts is regulated by the transcription factors, Runx2 and Osterix. Natural products of plant origin are still a major part of traditional medicinal systems in Korea. The root of Lithospermum erythrorhizon Sieb. et Zucc. (LR), the purple gromwell, is an herbal medicine used for inflammatory and infectious diseases. LR is an anti-inflammatory and exerts anticancer effects by inducing the apoptosis of cancer cells. However, the precise molecular signaling mechanisms of osteoblastogenesis as regards LR and osteoblast transcription are not yet known. In this study, we investigated the effects of ethanol (EtOH) extract of LR (LES) on the osteoblast differentiation of C2C12 myoblasts induced by bone morphogenetic protein 4 (BMP4) and the potential involvement of Runx2 and Osterix in these effects. We found that the LES exhibited an ability to induce osteoblast differentiation. LES increased the expression of the osteoblast marker, alkaline phosphatase (ALP), as well as its activity, as shown by ALP staining and ALP activity assay. LES also increased mineralization, as shown by Alizarin Red S staining. Treatment with LES increased the protein levels (as shown by immunoblotting), as well as the transcriptional activity of Runx2 and Osterix and enhanced osteogenic activity. These results suggest that LES modulates osteoblast differentiation at least in part through Runx2 and Osterix.

Estrogen receptor α (ER-α), which is involved in bone metabolism and breast cancer, has been shown to have transcriptional targets. Dlx3 is essential for the skeletal development and plays an important role in osteoblast differentiation. Various osteogenic stimulators and transcription factors can induce the protein expression of Dlx3. However, the regulatory function of ER-α in the Dlx3 mediated osteogenic process remains unknown. Therefore, we investigated the regulation of Dlx3 and found that ER-α is a positive regulator of Dlx3 transcription in BMP2-induced osteoblast differentiation. We also found that ER-α interacts with Dlx3 and increases its transcriptional activity and DNA binding affinity. Furthermore, we demonstrated that the regulation of Dlx3 activity by ER-α is independent of the ligand (estradiol) binding domain. These results indicate that Dlx3 is a novel target of ER-α, and that ER-α regulates the osteoblast differentiation through modulation of Dlx3 expression and/or interaction with Dlx3.

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.

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 differentiation in vitro and in 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 of κB 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-1β in 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.

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.

Interleukin (IL)-4 acts on T cells as a growth and activation factor, and promotes the differentiation of type 2 T helper cells. In T cells, expression of the gene encoding IL-4 is regulated by inducible or constitutive factors. Yin-Yang (YY)-1 is one of constitutive transcription factors binding to the IL-4 promoter. The recently identified YY2 protein is similar to YY1, with both sharing high levels of homology in their zinc finger motifs. However, the role of YY2 in T cells is unclear. YY1 and YY2 were constitutively expressed in EL4 T cells, and their expression was not dependent on stimulation. IL-4 promoter (-741/+56 fragment) activity was enhanced by YY1, but inhibited by YY2. The enhanced IL-4 promoter activity by YY1 was reduced by simultaneous expression of YY2. In addition, the DNA binding affinity of YY1 to the IL-4 promoter was adversely affected by YY2. Our results suggest that YY1 and YY2 exert opposing effects on the IL-4 promoter as they compete for the same DNA binding sites.

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.