WJCPR11 reverses the TNF-α-induced inhibition of adipocyte differentiation and glucose uptake.

Berberine is a natural isoquinoline alkaloid present in various herbs and is effective against metabolic syndrome in the pre-diabetic stage and high insulin resistance. The present study aimed to determine the effectiveness of WJCPR11, a berberine derivative that is commonly used for diabetes treatment, in ameliorating insulin resistance and diabetes treatment. WJCPR11 promoted adipocyte differentiation to a higher extent than other berberine derivatives and showed no noticeable toxicity in its effective concentration range. It increased the mRNA expression levels and protein abundance of adipogenic markers, including peroxisome proliferator-activated receptor γ (PPARγ), glucose transporter type 4 (GluT4), and fatty acid synthase (FAS), and markedly enhanced the level of adiponectin, a distinct marker of insulin sensitivity. Meanwhile, the mRNA levels of inflammatory markers such as plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6) were reduced after WJCPR11 treatment. Furthermore, the tumor necrosis factor-α (TNF-α)-induced inhibition of adipocyte differentiation and downregulation of glucose uptake were markedly reversed by WJCPR11 treatment. Collectively, the findings of this study indicate that WJCPR11 has great potential for diabetes treatment.

Stimulatory effects of platycodin D on osteoblast differentiation.

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 ß-catenin nuclear accumulation by upregulating GSK3ß phosphorylation. Furthermore, platycodin D upregulated the ALP activity and enhanced the mineralization process in osteoblast cells via the sirtuin 1/ß-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.

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.

Regulatory effects of 4-methoxychalcone on adipocyte differentiation through PPARγ activation and reverse effect on TNF-α in 3T3-L1 cells.

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.

Berberine bioisostere Q8 compound stimulates osteoblast differentiation and function in vitro.

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.

Platycodin D Inhibits Osteoclastogenesis by Repressing the NFATc1 and MAPK Signaling Pathway.

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.

Discovery of Isoquinolinoquinazolinones as a Novel Class of Potent PPARγ Antagonists with Anti-adipogenic Effects.

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.

Osterix represses adipogenesis by negatively regulating PPARγ transcriptional activity.

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.

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.

Estrogen Receptor α Regulates Dlx3-Mediated Osteoblast Differentiation.

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.

Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix.

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.

Src enhances osteogenic differentiation through phosphorylation of Osterix.

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.

Cbl regulates the activity of SIRT2.

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

Protein kinase A phosphorylates and regulates the osteogenic activity of Dlx5.

Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.

PKA-mediated stabilization of FoxH1 negatively regulates ERalpha activity.

Estrogen receptor alpha (ERalpha) mediates the mitogenic effects of estrogen. ERalpha signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled ERalpha activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent ERalpha activation, thereby facilitating ERalpha dimerization, promoter binding and coactivator recruitment. ERalpha can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several ERalpha-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses ERalpha transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate ERalpha, at least in part, through FoxH1.

Acetylation of Sirt2 by p300 attenuates its deacetylase activity.

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.