scholarly journals Lysyl Oxidase Down-Regulates The Osteoblastic Potential of BMP9 Through Inhibiting HIF-1α/Wnt/β-Catenin Axis in Mesenchymal Stem Cells

2021 ◽  
Author(s):  
Yan Deng ◽  
Honghong Luo ◽  
Yixuan Deng ◽  
Yuanyuan Yang ◽  
Xintong Yao ◽  
...  
Keyword(s):  
Bone Formation ◽  
Lysyl Oxidase ◽  
Mrna Level ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
Osteoblastic Differentiation ◽  
Histochemical Staining ◽  
Osteogenic Potential ◽  
Over Expression

Abstract Background: Bone morphogenetic protein 9 (BMP9) is one of excellent osteogenic factors, but it can also initiate adipogenesis concomitantly. Thus, the osteogenic potential of BMP9 may be enhanced if the adipogenesis were reduced. It was reported that lysyl oxidase (Lox) may function as a critical switcher for adipogenesis. Up to date, the role of Lox in BMP9-induced osteoblastic differentiation remains unknown.Methods: The effect and possible mechanism of Lox on the osteogenic function of BMP9 were evaluated with RT-PCR, western blotting, immunofluorescent and histochemical staining. The same results were also confirmed with the in vivo BMP9-induced ectopic bone formation model.Results: The mRNA and protein of Lox are both detectable in progenitor cells, and it was increased by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN and mineralization were all enhanced by inhibiting or silencing Lox, but reduced by exogenous Lox. BMP9 increased the mRNA level of c-Myc, which was enhanced by inhibiting Lox, so did the protein level of β-catenin. Effects of Lox specific inhibitor on BMP9-induced Runx2, OPN and mineralization were reduced obviously by silencing β-catenin. HIF-1α was up-regulated by BMP9, which was enhanced by inhibiting or silencing Lox, but decreased by Lox over-expression. The effects of Lox specific inhibitor on increasing BMP9-induced osteogenic markers were reduced greatly by silencing HIF-1α. On the contrary, the inhibitory effect of Lox on BMP9-induced osteoblastic markers was almost abolished by HIF-1α over-expression. BMP9-induced bone formation was increased by silencing Lox or over-expressing HIF-1α. The effect of silencing Lox on potentiating BMP9-induced bone formation was attenuated by silencing HIF-1α. Lox specific inhibitor increased the level of β-catenin and decreased that of SOST, but these effects were almost reversed by silencing HIF-1α.Conclusions: Lox may reducing the osteoblastic-induction function of BMP9 through inhibiting Wnt/β-catenin signal via down-regulation of HIF-1α partly.

scholarly journals In Vivo Study for Clinical Application of Dental Stem Cell Therapy Incorporated with Dental Titanium Implants

Materials ◽  
2021 ◽  
Vol 14 (2) ◽  
pp. 381
Author(s):  
Hyunmin Choi ◽  
Kyu-Hyung Park ◽  
Narae Jung ◽  
June-Sung Shim ◽  
Hong-Seok Moon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Alveolar Bone ◽  
Human Mesenchymal Stem Cells ◽  
Titanium Implants ◽  
Osteogenic Potential ◽  
Induction Medium ◽  
Radiographic Analysis ◽  
New Bone Formation

The aim of this study was to investigate the behavior of dental-derived human mesenchymal stem cells (d-hMSCs) in response to differently surface-treated implants and to evaluate the effect of d-hMSCs on local osteogenesis around an implant in vivo. d-hMSCs derived from alveolar bone were established and cultured on machined, sandblasted and acid-etched (SLA)-treated titanium discs with and without osteogenic induction medium. Their morphological and osteogenic potential was assessed by scanning electron microscopy (SEM) and real-time polymerase chain reaction (RT-PCR) via mixing of 5 × 106 of d-hMSCs with 1 mL of Metrigel and 20 μL of gel-cell mixture, which was dispensed into the defect followed by the placement of customized mini-implants (machined, SLA-treated implants) in New Zealand white rabbits. Following healing periods of 2 weeks and 12 weeks, the obtained samples in each group were analyzed radiographically, histomorphometrically and immunohistochemically. The quantitative change in osteogenic differentiation of d-hMSCs was identified according to the type of surface treatment. Radiographic analysis revealed that an increase in new bone formation was statistically significant in the d-hMSCs group. Histomorphometric analysis was in accordance with radiographic analysis, showing the significantly increased new bone formation in the d-hMSCs group regardless of time of sacrifice. Human nuclei A was identified near the area where d-hMSCs were implanted but the level of expression was found to be decreased as time passed. Within the limitations of the present study, in this animal model, the transplantation of d-hMSCs enhanced the new bone formation around an implant and the survival and function of the stem cells was experimentally proven up to 12 weeks post-sacrifice.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

scholarly journals The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Feng He ◽  
Zonghui Xiao ◽  
Hailan Yao ◽  
Sen Li ◽  
Miao Feng ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Cell Apoptosis ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
Mapk Signaling ◽  
Apoptosis Rate ◽  
Cvb3 Infection ◽  
Interfering Rna

Abstract Background The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. Methods We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. Results We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. Conclusions miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

4:51146. Nell-1 Induces Osteoblastic Differentiation of Bone Marrow Mesenchymal Cells In Vitro and Bone Formation In Vivo

2006 ◽  
Vol 6 (5) ◽  
pp. 74S
Author(s):  
Tara Aghaloo ◽  
Xinquan Jiang ◽  
Xinli Zhang ◽  
Zhang Zhiyuang ◽  
Jeffrey C. Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Osteoblastic Differentiation ◽  
Mesenchymal Cells ◽  
Bone Marrow Mesenchymal Cells

PTH and Bortezomib Suppress Growth of Primary Human Myeloma through Increased Bone Formation In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Angela Pennisi ◽  
Wen Ling ◽  
Paul Perkins ◽  
Rinku Saha ◽  
Xin Li ◽  
...  
Keyword(s):  
Bone Formation ◽  
Ex Vivo ◽  
Experimental Period ◽  
Inhibitory Effect ◽  
Tumor Burden ◽  
Anabolic Effect ◽  
X Rays ◽  
Mineral Density ◽  
Bortezomib Treatment

Abstract We have recently demonstrated the inhibitory effect of osteoblasts on myeloma (MM) ex vivo and in vivo (Yaccoby et al., Haematologica 2006) and that anti-MM response of bortezomib is associated with osteoblast activation in patients with MM (Zangari et al., BJH 2005). The aims of this study were to investigate the effect of intermittent PTH and bortezomib on bone remodeling and tumor growth, and the consequences of PTH pretreatment on MM progression in our SCID-rab model for primary MM (Yata & Yaccoby, Leukemia 2004). In nonmyelomatous hosts, both PTH and bortezomib significantly increased bone mineral density (BMD) of the implanted bone. SCID-rab mice were engrafted with MM cells from 13 patients. Following establishment of MM growth, as monitored by bi-weekly measurement of human monoclonal immunoglobulins (hIg) in mice sera and by x-rays, mice were injected subcutaneously with bortezomib (0.5 mg/kg twice a week, n=10), PTH (0.3 mg/kg/day, n=5) or PBS for 4–8 weeks. Whereas all PBS-treated mice had increased hIg levels during the experimental period, bortezomib treatment resulted in marked reduction of hIg in 5/10 experiments by 73±10% from pretreatment levels (responding hosts) and stabilized or delayed growth in additional 5 experiments. Overall, tumor burden in control- and bortezomib-treated mice was increased by 447±118% and 157±97% from pretreatment levels, respectively (p<0.02). Whereas in control mice the BMD of the implanted bone was reduced by 17±5% from pretreatment, it increased in bortezomib-treated hosts by 4±10% from pretreatment (p<0.03). While in bortezomib-responding hosts BMD increased by 20±14% (n=5), it decreased in partial/non-responding hosts (n=5) by 13±12% (n=5) from pretreatment (p<0.047). This bone anabolic effect was unique to bortezomib and was not observed in hosts responding to dexamethasone. Histological examination revealed that myelomatous bones from bortezomib-treated hosts had increased numbers of osteocalcin-expressing osteoblasts (34±7 vs. 13±3 per mm bone in control mice, p<0.03) and reduced numbers of multinucleated TRAP-expressing osteoclasts (10±3 vs. 28±7 per mm bone in control mice, p<0.02). We further demonstrated that bortezomib suppresses osteoclastogenesis through downregulation of NF-κB activity in osteoclast precursors. Since bortezomib also directly inhibits MM cell growth we further studied the association between increased bone formation and MM growth by treating hosts engrafted with MM cells from 5 patients with PTH, a bone anabolic agent with no known direct apoptotic effect on MM cells. Whereas PTH treatment resulted in increased BMD of the implanted bone by 19±5%, BMD in control hosts was reduced by 5±8% from pre-treatment (p<0.05). The bone anabolic effect of PTH was associated with inhibition of MM progression in 4/5 experiments. Overall, hIg in PBS- and PTH-treated mice was increased by 947±247% and 391±217% from pretreatment levels, respectively (p<0.04). In additional set of experiments hosts received PTH or PBS, 4 weeks prior to inoculation of MM cells from 3 patients and thereafter. In all experiments, PTH pretreatment, which increased implanted BMD by 48±11%, resulted in slower growth of MM cells as compared to control hosts. We conclude that increased bone formation by PTH and bortezomib contributes to controlling MM growth and that pretreatment with PTH, in addition to improving skeletal complications, may be a promising approach to prevent MM progression.

New targeted anti CDK4/6 peptide MM-D37K.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13545-e13545 ◽  
Author(s):  
Vladimir Konstantinovich Bozhenko ◽  
Tatyana Michailovna Kulinich ◽  
Elena Aleksandrovna Kudinova ◽  
Andrey Boldyrev ◽  
Vladimir Alekseevich Solodkij
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Full Range ◽  
Mrna Level ◽  
Xenograft Model ◽  
Specific Inhibitor ◽  
Antitumor Effects ◽  
Mcf 7

e13545 Background: MM-D37K is a synthetic peptide which consists of p16INK4a-specific inhibitor of complex cyclin D- CDK4 and CDK6 and cell penetrating peptide (CPP) – Antp (Penetratin). We investigated in vitro and in vivo cytotoxic, cytostatic and antitumor activity of MM-D37K. The level of cyclin A, Ki67,bax, bcl-2 and pRb phosphorylation was investigated. Full range of Toxicology tests and Pharmacokinetics experiments in mice, rats and rabbits were performed. Methods: Different cell lines (Jurcat, Raji, A549, MCF-7, Hct-116, Ht-29, HEK293) were incubated with 0.1-100 mM MM-D37K for 24-48 hrs. Proliferation (MTT), DNA-content, cell cycle (flow cytometry) and mRNA level of appropriate proteins (RT PCR) were investigated. In vivo experiments were conducted on xenograft model of HCT116, A-549 at concentration 5 and 10 mg/kg of MM-D37K. Toxicology experiments were made under RF Law and included 3 types of animals. LC-MS MMD37K method of detection in plasma was developed. Results: MM-D37K prevented pRb phosphorilation and proliferation activation in all investigated cell lines. Cell cycle was blocked in G1 phase. Cytostatic effect did not depend on p16 mutation or expression. MM-D37K induced apoptosis in 20-82% of investigated cells at 40 mM with lowest level for MCF-7. LD10 for rats was 100 mg/kg and no deaths were registered for rabbits (highest dose was 50 mg/kg). Concentration of MMD-37K in plasma after 2 min and bolus i.v. injection in dose 10 mg/kg was 72.16±5.64 mcg/ml. Concentration decreased in two phases. 1st – t1/2 = 2.39±0.39 min and for 2nd t1/2=2.39±0.39 hr. Antitumor effects in xenograft model were 53% for A-549 and 67% for HCT116. Conclusions: Our results proved cytotoxic, cytostatic and antitumor effects of MM-D37K in investigated cell lines in vitro and in vivo. Toxicological and pharmacokinetics results allow us recommend for I/IIa Phase clinical trial. (Support: MetaMax Ltd., RFFI, Minpromtorg RF.)

scholarly journals Involvement of Cyclic Adenosine 3′,5′-Monophosphate in the Differential Regulation of Activin βA and βB Expression by Gonadotropin in the Zebrafish Ovarian Follicle Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (2) ◽  
pp. 491-499 ◽  
Author(s):  
Yajun Wang ◽  
Wei Ge
Keyword(s):  
Oocyte Maturation ◽  
Ovarian Follicle ◽  
Mrna Level ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
Differential Regulation ◽  
Follicle Cells ◽  
Intracellular Camp ◽  
Dependent Manner

Activin is a dimeric protein consisting of two similar but distinct β-subunits, βA and βB. In our previous studies, both activin A (βAβA) and activin B (βBβB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin βA was cloned and sequenced. The precursor of zebrafish activin βA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin βA and βB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin βA-subunit; however, it significantly down-regulated the steady-state expression level of activin βB in a time- and dose-dependent manner. The differential regulation of the two β-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin βA expression at 10 μm, but it was unable to reverse the inhibitory effects of hCG and forskolin on βB expression. This suggests that the hCG-stimulated activin βA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin βB expression is likely mediated by PKA-independent pathway(s).

scholarly journals Increased BMSC Exosomal miR-140-3p Alleviates Bone Degradation and Promotes Bone Restoration by Targeting Plxnb1 in Diabetic Rats

2021 ◽  
Author(s):  
Ning Wang ◽  
Xuanchen Liu ◽  
Zhen Tang ◽  
Xinghui Wei ◽  
Hui Dong ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Bone Formation ◽  
Bone Regeneration ◽  
Bone Defect ◽  
Diabetic Rats ◽  
Osteogenic Potential ◽  
Bone Degradation ◽  
Defect Repair

Abstract Background: Diabetes mellitus (DM) is considered to be an important factor for bone degeneration disorders such as bone defect nonunion, which is characterized by physical disability and tremendous economy cost to families and society. Exosomal miRNAs of BMSCs have been reported to participate in osteoblastogenesis and modulating bone formation. However, their impacts on the development of bone degeneration in DM are not yet known. The role of miRNAs in BMSCs exosomes on regulating hyperglycemia bone degeneration was investigated in the present study. Results: The osteogenic potential in bone defect repair of exosomes derived from diabetes mellitus BMSCs derived exosomes (DM-Exos) were revealed to be lower than that in normal BMSCs derived exosomes (N-Exos) in vitro and in vivo. Here, we demonstrate that miR-140-3p level was significantly altered in exosomes derived from BMSCs, ADSCs and serum from DM rats. In in vitro experiments, upregulated miR-140-3p exosomes promoted DM BMSCs differentiation into osteoblasts. The effects were exerted by miR-140-3p targeting plxnb1, plexin B1 is the receptor of semaphoring 4D(Sema4D) that inhibited osteocytes differentiation, thereby promoting bone formation. In DM rats with bone defect, miR-140-3p upregulated exosomes were transplanted into injured bone and accelerated bone regeneration. Besides, miR-140-3p in the exosomes was transferred into BMSCs and osteoblasts and promoted bone regeneration by targeting the plexin B1/RohA/ROCK signaling pathway. Conclusions: Normal-Exos and miR-140-3p overexpressed-Exos accelerated diabetic wound healing by promoting the osteoblastogenesis function of BMSCs through inhibition plexin B1 expression which is the receptor of Sema4D and the plexin B1/RhoA/ROCK pathway compared with diabetes mellitus-Exos. This offers a new insight and a new therapy for treating diabetic bone unhealing.

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