Conditional knockout of the PDK‐1 gene in osteoblasts affects osteoblast differentiation and bone formation

2020 ◽  
Author(s):  
Yiguang Bai ◽  
Qiong Zhang ◽  
Qiaoling Chen ◽  
Quan Zhou ◽  
Yanan Zhang ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Conditional Knockout

scholarly journals Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation

2020 ◽  
Vol 295 (33) ◽  
pp. 11669-11681 ◽  
Author(s):  
Jun Tang ◽  
Jing Xie ◽  
Wei Chen ◽  
Chenyi Tang ◽  
Jinjin Wu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Bone Diseases ◽  
Conditional Knockout ◽  
Bone Homeostasis ◽  
Exact Function ◽  
Related Transcription Factor ◽  
Transcription Factor 1 ◽  
Osteoblast Maturation

Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 (Runx1) is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific Runx1 conditional knockout mouse model to study RUNX1's function in bone formation. Runx1 ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of Runx1f/fOsx-Cre mice compared with Runx1f/f and Osx-Cre mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the Runx2 gene promoter and up-regulates Runx2 expression. Furthermore, the ChIP data also showed that RUNX1 associates with the Ocn promoter. In conclusion, RUNX1 up-regulates the expression of Runx2 and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating Runx1 activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.

scholarly journals Deficiency of sphingomyelin synthase 1 but not sphingomyelin synthase 2 reduces bone formation due to impaired osteoblast differentiation

2019 ◽  
Vol 25 (1) ◽  
Author(s):  
Goichi Matsumoto ◽  
Chieko Hashizume ◽  
Ken Watanabe ◽  
Makoto Taniguchi ◽  
Toshiro Okazaki
Keyword(s):  
Golgi Apparatus ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Bone Development ◽  
Positive Control ◽  
Conditional Knockout ◽  
Mineral Apposition Rate ◽  
Mineral Density ◽  
Sphingomyelin Synthase

Abstract Background There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. Methods We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2−/− mouse). Results When we compared Sp7-Cre;SMS1f/f;SMS2−/− mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2−/−) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/− and Sp7-Cre;SMS1+/+;SMS2−/−), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2−/− mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/fSMS2−/− mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. Conclusions These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.

scholarly journals Kruppel-like factor 4 attenuates osteoblast formation, function, and cross talk with osteoclasts

2014 ◽  
Vol 204 (6) ◽  
pp. 1063-1074 ◽  
Author(s):  
Jung Ha Kim ◽  
Kabsun Kim ◽  
Bang Ung Youn ◽  
Jongwon Lee ◽  
Inyoung Kim ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Osteoclast Differentiation ◽  
Nuclear Factor Κb ◽  
Conditional Knockout ◽  
Mouse Bone Marrow ◽  
Bone Homeostasis ◽  
Rankl Expression

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)2D3-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)2D3-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.

scholarly journals Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 127-140 ◽  
Author(s):  
Shaohong Cheng ◽  
Weirong Xing ◽  
Sheila Pourteymoor ◽  
Jan Schulte ◽  
Subburaman Mohan
Keyword(s):  
Bone Formation ◽  
Growth Plate ◽  
Prolyl Hydroxylase ◽  
Conditional Knockout ◽  
Hypertrophic Chondrocytes ◽  
Endochondral Bone Formation ◽  
Bone Surface ◽  
Endochondral Bone ◽  
Prolyl Hydroxylase Domain

Abstract The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

OP0111 Mechanism of New Bone Formation in Ankylosing Spondylitis: the Role of IL-32 in Osteoblast Differentiation

2014 ◽  
Vol 73 (Suppl 2) ◽  
pp. 103.1-103
Author(s):  
S. Hong ◽  
E.-J. Lee ◽  
Y.J. Kim ◽  
B.S. Koo ◽  
E.-J. Chang ◽  
...  
Keyword(s):  
Ankylosing Spondylitis ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
New Bone Formation

scholarly journals miR-16-2* Interferes with WNT5A to Regulate Osteogenesis of Mesenchymal Stem Cells

2018 ◽  
Vol 51 (3) ◽  
pp. 1087-1102 ◽  
Author(s):  
Lijun Duan ◽  
He Zhao ◽  
Yang Xiong ◽  
Xiangsheng Tang ◽  
Yongdong Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Fragility Fractures ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Qrt Pcr ◽  
Wnt Signal

Background/Aims: Osteoporosis is a bone metabolic disease characterized by a systemic impairment of bone mass, which results in increased propensity of fragility fractures. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. Mesenchymal stem cells (MSCs) are induced to differentiate into preosteoblasts, which are regulated by the signaling cascades initiated by the various signals, including miRNAs. miR-16-2* is a newly discovered miRNA that participates in diagnosis and prognosis of hepatocellular carcinoma, cervical cancer and chronic lymphocytic leukemia. However, the effect of miR-16-2* on the regulation of osteoblast differentiation and the mechanism responsible are still unclear. Here we discuss the contribution of miR-16-2* to osteoporosis, osteoblast differentiation and mineralization. Methods: The expression pattern of miR-16-2* during osteogenesis or in osteoporosis bone samples was validated by quantitative real-time PCR (qRT-PCR). The human bone marrow mesenchymal stem cells (hBMSCs) were induced to differentiate into osteoblasts by osteogenic induced medium containing dexamethasone, ascorbate-2-phosphat, beta-glycerophosphate and vitamin-D3. The target genes of miR-16-2* were predicted by TargetScan and PicTar. The mRNA and protein levels of osteogenic key markers were detected using qRT-PCR or western blot respectively. The WNT signal activity was analyzed by TOP/FOP reporter assay. Results: The expression of miR-16-2* in patient bone tissue with osteoporosis was negatively correlated with bone formation related genes. During osteoblast differentiation process, the expression of miR-16-2* was significantly decreased. Upregulation of miR-16-2* in hBMSCs impaired the osteogenic differentiation while the downregulation of miR-16-2* increased this process. Upregulation the expression of miR-16-2* could also block the WNT signal pathway by directly target WNT5A. Furthermore, knockdown of miR-16-2* could promote the activation of RUNX2, possibly by lifting the inhibitory effect of miR-16-2* on WNT pathway. Conclusion: Taken together, we report a novel biological role of miR-16-2* in osteogenesis through regulating WNT5A response for the first time. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.

scholarly journals Effects of Farnesyl Pyrophosphate Accumulation on Calvarial Osteoblast Differentiation

Endocrinology ◽  
2011 ◽  
Vol 152 (8) ◽  
pp. 3113-3122 ◽  
Author(s):  
Megan M. Weivoda ◽  
Raymond J. Hohl
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Biosynthetic Pathway ◽  
Squalene Synthase ◽  
Farnesyl Pyrophosphate ◽  
Matrix Mineralization ◽  
Lower Serum Cholesterol ◽  
Calvarial Osteoblast ◽  
Isoprenoid Biosynthetic Pathway

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts. Interestingly, the effects of FPP accumulation on osteoblasts were found to be independent of protein farnesylation. Our findings are the first to demonstrate that the accumulation of FPP impairs osteoblast differentiation and suggests that the depletion of this isoprenoid may be necessary for normal and statin-induced bone formation.

scholarly journals The Effect of the Combination of Vitamin K2 and Genistein, Coumestrol and Daidzein on the Osteoblast Differentiation and Bone Matrix Formation

2016 ◽  
Vol 10 (2) ◽  
pp. 12-19
Author(s):  
Sahar S. Karieb ◽  
Mohammed M. Jawad ◽  
Hanady S. Al-Shmgani ◽  
Zahraa H.M. Kadri
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Nuclear Factor Kappa B ◽  
Type I Collagen ◽  
Bone Mineralization ◽  
Bone Matrix ◽  
Vitamin K2 ◽  
Type I ◽  
Nuclear Factor ◽  
Effective Factor

Multiple studies have been reported the stimulatory effect of the combinations of nutrients factors on bone formation. One such factor is vitamin K2 which can be associated with bone protective activities. The effect of vitamin K2 alone and in combination with genistein, coumestrol and daidzein on osteoblast differentiation and mineralization were tested. Significantly, vitamin K2 increased bone mineralization in combination with genistein (10-5M), coumestrol (10-7M) and daidzein (10-5M). However, there is no additive effect of this vitamin on alkaline phosphatase (ALP) levels in osteoblasts. By contrast, vitamin K2 enhanced the stimulatory effect of type I collagen and osteocalcin expression. Vitamin K2 alone increased RUNX and OSX expression while there is no synergistic effect with tested compound; this vitamin also did not modulate nuclear factor kappa B ligand (RANKL)/ osteoprotegerin (OPG) ratio expression. These results suggested that vitamin K2 can be more effective factor in the presence of phytoestrogens on the improvement of bone formation after menopause.

Osteoblast differentiation and bone formation are retarded in hypothermia

Bone ◽  
2010 ◽  
Vol 47 ◽  
pp. S129-S130
Author(s):  
J.J. Patel ◽  
D. Talbot ◽  
I.R. Orriss ◽  
M.L. Key ◽  
S.E.B. Taylor ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation

IL-3 promotes osteoblast differentiation and bone formation in human mesenchymal stem cells

2012 ◽  
Vol 418 (4) ◽  
pp. 669-675 ◽  
Author(s):  
Amruta P. Barhanpurkar ◽  
Navita Gupta ◽  
Rupesh K. Srivastava ◽  
Geetanjali B. Tomar ◽  
Sameer P. Naik ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Human Mesenchymal Stem Cells
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