YY2 Promotes Osteoblast Differentiation by Upregulating Osterix Transcriptional Activity.

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.

Pseudoshikonin I enhances osteoblast differentiation by stimulating Runx2 and Osterix.

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

Ethanol extract of Lithospermum erythrorhizon Sieb. et Zucc. promotes osteoblastogenesis through the regulation of Runx2 and Osterix.

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.

Yin-Yang 1 and Yin-Yang 2 exert opposing effects on the promoter activity of interleukin 4.

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.

Yin Yang 1 is a multi-functional regulator of adipocyte differentiation in 3T3-L1 cells.

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/EBPß (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/EBPß 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.

Smurf2 regulates the degradation of YY1.

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.

YY1 represses the transcriptional activity of Runx2 in C2C12 cells.

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.

The laxative effects of Maesil (Prunus mume Siebold & Zucc.) on constipation induced by a low-fibre diet in a rat model.

Maesil (the fruit of Prunus mume Siebold & Zucc.) has long been used as an alternative medicine and functional food in Korea and Japan for preventive and therapeutic purposes. We examined the laxative effect of unripe Maesil (UM) and ripe Maesil (RM) in a rat model on constipation induced by a low-fibre diet and the possible mechanisms of Maesil in the rat colon. In vivo studies were conducted on the low-fibre diet-induced constipation rat model, and isolated rat colon was used in in vitro experiments to measure the changes in spontaneous colon contraction generated by Maesil and organic acids as standard and effectual ingredients, respectively. The aqueous extract of both UM and RM applied orally (100 and 300 mg/kg) produced significant increase of faeces frequency (p < 0.05) and moisture (p < 0.001). Moreover, the number faecal pellets number was reduced (p < 0.05) in the distal colons of the Maesil-treated rats. Gastrointestinal (GI) motility, measured by charcoal meal, was activated more fully by UM than in the low-fibre diet group. Both UM and RM and its organic acids produced a dose-dependent stimulation of the spontaneous contractile amplitude (p < 0.001) and frequency (p < 0.01) of the isolated rat colon. Although both UM and RM were an effective laxative, the RM was significantly more effective than the UM in the in vivo and in vitro constipation experiments because of the changes in the composition of organic acids during the ripening of the fruit. Our results demonstrated that Maesil was effective in promoting the frequency of defaecation and contraction of the rat colon, which provided scientific basis to support the use of Maesil as potential therapeutics in treating constipation.

Saponins from the roots of Platycodon grandiflorum stimulate osteoblast differentiation via p38 MAPK- and ERK-dependent RUNX2 activation.

Changkil (CK), the aqueous extract of the roots of Platycodon grandiflorum, has been used as a traditional oriental medicine for the treatment of chronic adult diseases. Although a saponin fraction derived from CK (CKS) has been suggested to have a variety of functional effects, its effect on bone is unknown. In the present study, the effects of CKS on osteoblast differentiation and function were determined by analyzing the activity of alkaline phosphatase (ALP), an osteoblast marker, and the regulation of RUNX2, a master gene of osteoblast differentiation, in a mesenchymal stem cell line. CKS upregulated ALP activity and the expression of osteogenic marker genes in C2C12 cells. In addition, CKS increased the expression and transcriptional activity of RUNX2. To determine which signaling pathways are involved in the osteogenic effects of CKS, we tested the effect of inhibitors of kinases known to regulate RUNX2. CKS-induced enhancement of RUNX2 and ALP was inhibited by treatment with a p38 inhibitor (SB203580) and an ERK inhibitor (U0126). These findings suggest that CKS stimulates osteoblast differentiation by activation of RUNX2 via mechanisms related to the p38 MAPK and ERK signaling pathways. The regulation of RUNX2 activation by CKS may be an important therapeutic target for osteoporosis.

SIRT1 inhibitory diterpenoids from the Vietnamese medicinal plant Croton tonkinensis.

Silent information regulator two ortholog 1 (SIRT1) is a member of the sirtuin deacetylase family of enzymes that removes acetyl groups from the lysine residues in histones and other proteins. It has been suggested that SIRT1 inhibitors might be beneficial in the treatment of cancer and neurodegenerative diseases. Bioassay-guided fractionation of the MeOH extract of the leaves of CROTON TONKINENSIS resulted in the isolation of a new ENT-kaurane diterpenoid (1) along with 11 known compounds (2- 12). The structure of the new compound 1 was determined to be ENT-11 alpha-acetoxy-7 beta-hydroxykaur-16-en-15-one based on spectroscopic analyses. Compounds 3, 4, 6- 9, 11, and 12 exhibited SIRT1 inhibitory activity in an IN VITRO assay, with IC (50) values ranging from 16.08 +/- 0.11 to 44.34 +/- 2.32 microM. This is the first report showing the potential of ENT-kaurane diterpenoids as a new class of natural SIRT1 inhibitors.

Crotonkinensins A and B, diterpenoids from the Vietnamese medicinal plant Croton tonkinensis.

Two new diterpenoids, crotonkinensins A (1) and B (2), were isolated from the leaves of the Vietnamese endemic medicinal plant Croton tonkinensis. Their structures were determined to be 7alpha,10alpha-epoxy-14beta-hydroxygrayanane-1(5),16(17)-dien-2,15-dione (1) and 7alpha,10alpha-epoxy-14beta-hydroxygrayanane-1(2),16(17)-dien-15-one (2) by spectroscopic analysis. Compounds 1 and 2 showed strong anti-inflammatory effects on the LPS-induced COX-2 promoter activity and COX-2 expression in Raw 264.7 cells.

Inhibition of interleukin-2 production by myricetin in mouse EL-4 T cells.

Myricetin is a naturally occurring flavonoid that is commonly found in tea, berries, fruits, vegetables, and medicinal herbs. This study examined the effects of myricetin on the production of interlukin-2 (IL-2), a potent T cell growth factor. Treatment with myricetin significantly inhibited the secretion of the IL-2 protein from mouse EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) in a dose-dependent manner. Flow cytometric analysis showed that myricetin suppressed the intracellular production of the IL-2 protein. Furthermore, the effects of myricetin on mRNA expression were analyzed by reverse transcription-polymerase chain reaction and it showed that myricetin reduced the expression of IL-2 mRNA induced by PMA plus Io. This suggests that myricetin has potential immunosuppressive effects by inhibiting the production of IL-2.