Macrophage-specific FGF12 promotes liver fibrosis progression in mice.

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: FGF12 was up-regulated in bile duct ligation (BDL)-induced and CCL 4 -induced liver fibrosis mouse models. Expression of FGF12 was specifically up-regulated in nonparenchymal liver cells, especially in hepatic macrophages. By constructing myeloid-specific FGF12 knockout mice, we found that deletion of FGF12 in macrophages protected against BDL-induced and CCL 4 -induced liver fibrosis. Further results revealed that FGF12 deletion dramatically decreased the population of lymphocyte antigen 6 complex locus C high macrophages in mouse fibrotic liver tissue and reduced the expression of proinflammatory cytokines and chemokines. Meanwhile, loss-of-function and gain-of-function approaches revealed that FGF12 promoted the proinflammatory activation of macrophages, thus inducing HSC activation mainly through the monocyte chemoattractant protein-1/chemokine (C-C motif) receptor 2 axis. Further experiments indicated that the regulation of macrophage activation by FGF12 was mainly mediated through the Janus kinase-signal transducer of activators of transcription pathway. Finally, the results revealed that FGF12 expression correlates with the severity of fibrosis across the spectrum of fibrogenesis in human liver samples. CONCLUSIONS: FGF12 promotes liver fibrosis progression. Therapeutic approaches to inhibit macrophage FGF12 may be used to combat liver fibrosis in the future.

Macrophage-specific FGFR1 deletion alleviates high-fat-diet-induced liver inflammation by inhibiting the MAPKs/TNF pathways.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease that is substantially associated with obesity-induced chronic inflammation. Macrophage activation and macrophage-medicated inflammation play crucial roles in the development and progression of NAFLD. Furthermore, fibroblast growth factor receptor 1 (FGFR1) has been shown to be essentially involved in macrophage activation. This study investigated the role of FGFR1 in the NAFLD pathogenesis and indicated that a high-fat diet (HFD) increased p-FGFR1 levels in the mouse liver, which is associated with increased macrophage infiltration. In addition, macrophage-specific FGFR1 knockout or administration of FGFR1 inhibitor markedly protected the liver from HFD-induced lipid accumulation, fibrosis, and inflammatory responses. The mechanistic study showed that macrophage-specific FGFR1 knockout alleviated HFD-induced liver inflammation by suppressing the activation of MAPKs and TNF signaling pathways and reduced fat deposition in hepatocytes, thereby inhibiting the activation of hepatic stellate cells. In conclusion, the results of this research revealed that FGFR1 could protect the liver of HFD-fed mice by inhibiting MAPKs/TNF-mediated inflammatory responses in macrophages. Therefore, FGFR1 can be employed as a target to prevent the development and progression of NAFLD.

Deubiquitinase USP17 Regulates Osteoblast Differentiation by Increasing Osterix Protein Stability.

Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, USP17 overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, Usp17 knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of Usp17 led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of USP17 led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts.

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.

Cyclophilin A Promotes Osteoblast Differentiation by Regulating Runx2.

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.

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.

Microbial Transformation of Broussochalcones A and B by Aspergillus niger.

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.

Dlx5 Represses the Transcriptional Activity of PPARγ.

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.

Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress.

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.

Synergistic stimulating effect of 2-hydroxymelatonin and BMP-4 on osteogenic differentiation in vitro.

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.

Protective effect of EX-527 against high-fat diet-induced diabetic nephropathy in Zucker rats.

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-ß1 (49%), interleukin (IL)-1ß (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-ß (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-ß, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

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.

SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development.

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.

Estrogen Deficiency Potentiates Thioacetamide-Induced Hepatic Fibrosis in Sprague-Dawley Rats.

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 γ-glutamyl transferase (GGT) were significantly increased compared to those in TAA-treated sham-operated OVX rats or TAA-treated male rats. Furthermore, α-smooth muscle actin (α-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-α (TNF-α) concentrations were exhibited in TAA-treated OVX rats, and this further aggravated fibrosis through the transforming growth factor-ß (TGF-ß)/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.

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.

Berberine derivative, Q8, stimulates osteogenic 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 ß-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.

Hepatic damage exacerbates cisplatin-induced acute kidney injury in Sprague-Dawley rats.

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.

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.

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.

Osterix plays a critical role in BMP4-induced promoter activity of connexin43.

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 overexpression 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.