CREB/Wnt10b mediates the effect of COX-2 on promoting BMP9-induced osteogenic differentiation via reducing adipogenic differentiation in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic factors, which may be a potential candidate for bone tissue engineering. However, the osteogenic capacity of BMP9 still need to be further enhanced. In this study, we determined the effect of Wnt10b on BMP9-induced osteogenic differentiation in mesenchymal stem cell (MSCs) and the possible mechanism underlying this process. We introduced the polymerase chain reaction (PCR), Western blot analysis, histochemical stain, ectopic bone formation, and microcomputed tomography analysis to evaluate the effect of Wnt10b on BMP9-induced osteogenic differentiation. Meanwhile, PCR, Western blot analysis, chromatin immunoprecipitation, and immunoprecipitation were used to analyze the possible relationship between BMP9 and Wnt10b. We found that BMP9 upregulates Wnt10b in C3H10T1/2 cells. Wnt10b increases the osteogenic markers and bone formation induced by BMP9 in C3H10T1/2 cells, and silencing Wnt10b decreases these effects of BMP9. Meanwhile, Wnt10b enhances the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) induced by BMP9, which can be reduced by silencing Wnt10b. On the contrary, Wnt10b inhibits adipogenic markers induced by BMP9, which can be decreased by silencing Wnt10b. Further analysis indicated that BMP9 upregulates cyclooxygenase-2 (COX-2) and phosphorylation of cAMP-responsive element binding (p-CREB) simultaneously. COX-2 potentiates the effect of BMP9 on increasing p-CREB and Wnt10b, while silencing COX-2 decreases these effects. p-CREB interacts with p-Smad1/5/8 to bind the promoter of Wnt10b in C3H10T1/2 cells. Our findings suggested that Wnt10b can promote BMP9-induced osteogenic differentiation in MSCs, which may be mediated through enhancing BMP/Smad signal and reducing adipogenic differentiation; BMP9 may upregulate Wnt10b via the COX-2/p-CREB-dependent manner.

Wnt11 promotes BMP9-induced osteogenic differentiation through BMPs/Smads and p38 MAPK in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9), as one of the most potent osteogenic factors, is a promising cytokine for bone tissue engineering. Wnt11 can regulate the development of the skeletal system and is related to high bone mass syndrome. However, the effect of Wnt11 on BMP9-induced osteogenic differentiation remains unknown. In this study, we investigated the relationship between Wnt11- and BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs). We recapitulated the osteogenic potential of BMP9 in C3H10T1/2 cells. The messenger RNA expression of Wnt11 is detectable in the available progenitor cells, and BMP9 can obviously increase the protein level of Wnt11 in these cells. Exogenous Wnt11 potentiates the effect of BMP9 on increasing alkaline phosphatase (ALP) activities, the expression of osteopontin (OPN), and Runt-related transcription factor 2 (Runx2), so does matrix mineralization in C3H10T1/2 cells. Although Wnt11 cannot increase the BMP9-induced ectopic bone formation, it can increase the bone density induced by BMP9 apparently. Wnt11 increases the level of p-Smad1/5/8, as well as p-p38. Meanwhile, Wnt11 promotes the effect of BMP9 on increasing the levels of p-Smad1/5/8 and p-p38. Inhibition of p38 decreases the BMP9-induced ALP activities, the expression of OPN, and the mineralization in C3H10T1/2 cells. However, all of these effects of the p38 inhibitor on BMP9-induced osteogenic markers can be almost reversed by the overexpression of Wnt11. Our findings suggested that Wnt11 can enhance the osteogenic potential of BMP9 in MSCs, and this effect may be partly mediated through enhancing BMPs/Smads and the p38 MAPK signal, which was induced by BMP9.

BMP9/COX-2 axial mediates high phosphate-induced calcification in vascular smooth muscle cells via Wnt/ß-catenin pathway.

Vascular calcification is a notable risk factor for cardiovascular system. High phosphate can induce calcification in vascular smooth muscle cells (VSMCs), but the detail mechanism underlying this process remains unclear. In the present study, we determined the relationship between high phosphate and bone morphogenetic protein 9 (BMP9) in VSMCs, the effect of BMP9 on calcification in VSMCs and the effect of COX-2 on BMP9 induced calcification in VSMCs, as well as the possible mechanism underlying this biological process. We found that high phosphate obviously up-regulates the expression of BMP9 in VSMCs. Over-expression of BMP9 decreases the level of alpha-smooth muscle cell actin (α-SMA) apparently, but increases the level of Runx-2, Dlx-5, and ALP in VSMCs. Meanwhile, BMP9 increases the level of OPN and OCN, promotes mineralization in VSMCs and induces calcification in thoracic aorta. High phosphate and over-expression of BMP9 increases the level of COX-2. Over-expression of COX-2 enhances the inhibitory effect of BMP9 on α-SAM and increases the level of OPN and OCN induced by BMP9. However, inhibition of COX-2 decreases the BMP9-induced calcification in VSMCs and thoracic aorta. For mechanism, we found that high phosphate or BMP9 increases the level of ß-catenin and p-GSK3ß in VSMCs, but no substantial effect on GSK3ß. However, COX-2 inhibitor decreases the expression of ß-catenin induced by BMP9. Our findings indicated that BMP9 is involved in the phosphate-induced calcification in VSMCs and COX-2 partly mediates the BMP9-induced calcification in VSMCs through activating Wnt/ß-catenin pathway.

[Analysis on traditional Chinese medicine prescriptions treating cancer based on traditional Chinese medicine inheritance assistance system and discovery of new prescriptions].

Malignant tumor is one of the main causes for death in the world at present as well as a major disease seriously harming human health and life and restricting the social and economic development. There are many kinds of reports about traditional Chinese medicine patent prescriptions, empirical prescriptions and self-made prescriptions treating cancer, and prescription rules were often analyzed based on medication frequency. Such methods were applicable for discovering dominant experience but hard to have an innovative discovery and knowledge. In this paper, based on the traditional Chinese medicine inheritance assistance system, the software integration of mutual information improvement method, complex system entropy clustering and unsupervised entropy-level clustering data mining methods was adopted to analyze the rules of traditional Chinese medicine prescriptions for cancer. Totally 114 prescriptions were selected, the frequency of herbs in prescription was determined, and 85 core combinations and 13 new prescriptions were indentified. The traditional Chinese medicine inheritance assistance system, as a valuable traditional Chinese medicine research-supporting tool, can be used to record, manage, inquire and analyze prescription data.

Pyruvate dehydrogenase kinase 4 promotes osteoblastic potential of BMP9 by boosting Wnt/ß-catenin signaling in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is an effective osteogenic factor and a promising candidate for bone tissue engineering. The osteoblastic potential of BMP9 needs to be further increased to overcome its shortcomings. However, the details of how BMP9 triggers osteogenic differentiation in mesenchymal stem cells (MSCs) are unclear. In this study, we used real-time PCR, western blot, histochemical staining, mouse ectopic bone formation model, immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in BMP9-induced osteogenic differentiation of C3H10T1/2 cells, as well as the underlying mechanism. We found that PDK4 was upregulated by BMP9 in C3H10T1/2 cells. BMP9-induced osteogenic markers and bone mass were increased by PDK4 overexpression, but decreased by PDK4 silencing. ß-catenin protein level was increased by BMP9, which was enhanced by PDK overexpression and decreased by PDK4 silencing. BMP9-induced osteogenic markers were reduced by PDK4 silencing, which was almost reversed by ß-catenin overexpression. PDK4 increased the BMP9-induced osteogenic markers, which was almost eliminated by ß-catenin silencing. Sclerostin was mildly decreased by BMP9 or PDK4, and significantly decreased by combined BMP9 and PDK4. In contrast, sclerostin increased significantly when BMP9 was combined with PDK4 silencing. BMP9-induced p-SMAD1/5/9 was increased by PDK4 overexpression, but was reduced by PDK4 silencing. PDK4 interacts with p-SMAD1/5/9 and regulates the sclerostin promoter. These findings suggest that PDK4 can increase the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via the downregulation of sclerostin. PDK4 may be an effective target to strengthen BMP9-induced osteogenesis.

Cyclooxygenase-2/sclerostin mediates TGF-ß1-induced calcification in vascular smooth muscle cells and rats undergoing renal failure.

In this study, we studied the effect and possible mechanism of TGF-ß1 on vascular calcification. We found that the serum levels of TGF-ß1 and cycloxygenase-2 (COX-2) were significantly increased in patients with chronic kidney disease. Phosphate up regulated TGF-ß1 in vascular smooth muscle cells (VSMCs). TGF-ß1 decreased the markers of VSMCs, but increased osteogenic markers and calcification in aortic segments. The phosphate-induced osteogenic markers were reduced by the TGFßR I inhibitor (LY364947), which also attenuated the potential of phosphate to reduce VSMC markers in VSMCs. Both phosphate and TGF-ß1 increased the protein level of ß-catenin, which was partially mitigated by LY364947. TGF-ß1 decreased sclerostin, and exogenous sclerostin decreased the mineralization induced by TGF-ß1. LY364947 reduced the phosphate and TGF-ß1 induced COX-2. Meanwhile, the effects of TGF-ß1 on osteogenic markers, ß-catenin, and sclerostin, were partially reversed by the COX-2 inhibitor. Mechanistically, we found that p-Smad2/3 and p-CREB were both enriched at the promoter regions of sclerostin and ß-catenin. TGF-ß1 and COX-2 were significantly elevated in serum and aorta of rats undergoing renal failure. Therapeutic administration of meloxicam effectively ameliorated the renal lesion. Our results suggested that COX-2 may mediate the effect of TGF-ß1 on vascular calcification through down-regulating sclerostin in VMSCs.

BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling.

Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.

[Effect of BMP-2 and -3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertebral disc cells in vitro].

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein (BMP)-2 and -3 over expressions on chondrogenesis and osteogenesis of prenatal mouse intervertebral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases. METHODS: The prenatal mouse intervertebral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I (Col I), collagen type II (Col II), aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT-PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase (ALP) activity was detected with ALP reading and ALP staining. RESULTS: BMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus (AF) cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus (NP) cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP-2, but not BMP-3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP-2 and BMP-3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells. CONCLUSION: Adenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.

[Soft tissue reconstruction for secondary deformity after correction of Wassel type IV-D thumb duplication].

OBJECTIVE: To investigate an effective therapeutic method for the secondary deformity after the correction of the Wassel type IV thumb duplication. METHODS: 9 cases of Wassel W-D Complex thumb deformities in children with postoperative secondary deformity, including 6 males and 3 female, were treated. The age ranged from 2.0 to 14 years old with an average of 5.3 years old. During the operation, the anatomical structure was dissected to observe the structure and alignment of the flexor tendon as well as anatomical structure of the joint. In the meantime, the flexor pollicis longus tendon was shifted, A2 pulley was reconstructed, joint capsule was released and contracted, the end point of thenar was shifted. Kirschner wires fixation were used for about 4-5 weeks, the brace fixation for about 3 months. RESULTS: All the patients had radial side skin contracture of the interphalangeal joint, radial deviation of the thumb tip, radial side contracture and ulnar relaxation of the joint capsule. Flexor hallucis longus tendon was located in front of the radial side of the proximal phalanx, with no wrapped sheath or A2 pulley. Flexor hallucis longus tendon was attached to the thumb tip substrate, of which 1/3 was located in the center and 2/3 in the radial side. The thumb tip rotated about 10 degrees-15 degrees to the radial side. The patients were followed up for 6-38 months, with an average of 24 months. We adopted Tada standard to evaluate the follow-up results as excellent in 7 cases, good in 1 case, poor in 1 case. CONCLUSIONS: Soft tissue reconstruction for the secondary deformity after the correction of the Wassel type IV-D thumb duplication is an effective method. Application of the brace after removal of Kirschner wires has an important role in preventing the secondary deformity.