BMP2
Bone morphogenetic protein 2 precursor (BMP-2) (Bone morphogenetic protein 2A) (BMP-2A) [BMP2A]
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- Adipose-derived stem cells (ADSCs) are ideal for cell-based therapies to support bone regeneration. It is vital to understand the critical genes and molecular mechanisms involved in the functional regulation of ADSCs for enhancing bone regeneration. In the present study, we investigated the Gremlin 1 (GREM1) effect on ADSCs osteogenic differentiation and senescence. : The [i]in vitro[/i] ADSCs osteogenic differentiation potential was evaluated by determining alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteogenic markers. Cell senescence is determined by SA-β-gal staining, telomerase assay, and the expression of aging markers. : GREM1 overexpression in ADSCs reduced ALP activity and mineralization, inhibited the expression of osteogenic related genes [i]OCN, OPN, DSPP, DMP1[/i], and [i]BSP[/i], and key transcription factors, [i]RUNX2[/i] and [i]OSX[/i]. GREM1 knockdown in ADSCs enhanced ALP activity and mineralization, promoted the expression of [i]OCN, OPN, DSPP, DMP1, BSP, RUNX2[/i], and [i]OSX[/i]. GREM1 overexpression in ADSCs reduced the percent SA-β-Gal positive cells, [i]P16[/i] and [i]P53[/i] expressions, and increased telomerase activity. GREM1 knockdown in ADSCs increased the percentage of SA-β-Gal positive cells, [i]P16[/i] and [i]P53[/i] expressions, and reduced telomerase activity. Furthermore, GREM1 reduced the mRNA expression levels of BMP2, BMP6, and BMP7. : In summary, our findings suggested that GREM1 inhibited ADSCs senescence and osteogenic differentiation and antagonized BMP transcription.
Keywords
- BMP
- GREM1
- adipose-derived stem cells (ADSCs)
- osteogenic differentiation
- senescence
Interleukin (IL)-1β, as a key biomarker and mediator of vascular calcification in patients with end-stage renal disease (ESRD), may be involved in the process of premature senescence of vascular smooth muscle cells (VSMCs). This work sought to investigate whether IL-1β-induced premature senescence contributes to the process of osteoblastic transition and vascular calcification in VSMCs. Eighty-eight patients with ESRD (aged 25-81 years), 11 healthy individuals, and 15 cases of lesion-free distal radial arteries from dialysis ESRD patients with angiostomy were collected in this study. Immunohistochemical analysis was performed to detect expression of IL-1β, p21, and bone morphogenetic protein-2 (BMP2) in the distal radial arteries. Primary human VSMCs from healthy neonatal umbilical cords were incubated with test agents for 1-3 days. Intracellular levels of reactive oxygen species (ROS) and senescence-associated-β-galactosidase (SA-β-gal) staining were used to detect senescent cells. Alizarin red staining and the calcium content of the cell layer were used to detect mineral deposition in VSMCs. Coincident with positive staining of IL-1β, p21, and BMP2 in the lesion-free distal radial arteries, 66.67% patients showed mineral deposition. Serum IL-1β was 0.24 ± 0.57, 1.20 ± 2.95, and 9.41 ± 40.52 pg/mL in 11 healthy individuals, 20 patients without calcification, and 53 patients with calcification, respectively. Analysis of the cross-table chi-square test showed cardiovascular calcification is not correlated with levels of serum IL-1β in patients with ESRD (p = 0.533). In response to IL-1β, VSMCs showed a senescence-like phenotype, such as flat and enlarged morphology, increased expression of p21, an increased activity of SA-β-gal, and increased levels of ROS. IL-1β-induced senescence of VSMCs was required for the activation of IL-1β/NF-κB/p53/p21 signaling pathway. IL-1β-induced senescent VSMCs underwent calcification due to osteoblastic transition mainly depending upon the upregulation of BMP2. Resveratrol, an activator of sirtuin-1, postponed the IL-1β-induced senescence through blocking the NF-κB/p53/p21 pathway and attenuated the osteoblastic transition and calcification in VSMCs. High levels of IL-1β in medial smooth muscles of arteries may play roles in inducing senescence-associated calcification. IL-1β-induced senescence depending on the activation of the NF-κB/p53/p21 signaling pathway and contributing to osteoblastic transition of VSMCs.
MeSH Terms
- Adult
- Aged
- Aged, 80 and over
- Female
- Humans
- Interleukin-1beta
- Male
- Middle Aged
- Muscle, Smooth, Vascular
- Osteoblasts
Keywords
- Interleukin-1β
- Osteoblastic transition
- Senescence
- Vascular calcification
Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-related lineage switch between osteogenic and adipogenic fates, which contributes to bone loss and adiposity. Here we identified a long noncoding RNA, Bmncr, which regulated the fate of BMSCs during aging. Mice depleted of Bmncr (Bmncr-KO) showed decreased bone mass and increased bone marrow adiposity, whereas transgenic overexpression of Bmncr (Bmncr-Tg) alleviated bone loss and bone marrow fat accumulation. Bmncr regulated the osteogenic niche of BMSCs by maintaining extracellular matrix protein fibromodulin (FMOD) and activation of the BMP2 pathway. Bmncr affected local 3D chromatin structure and transcription of Fmod. The absence of Fmod modified the bone phenotype of Bmncr-Tg mice. Further analysis revealed that Bmncr would serve as a scaffold to facilitate the interaction of TAZ and ABL, and thus facilitate the assembly of the TAZ and RUNX2/PPARG transcriptional complex, promoting osteogenesis and inhibiting adipogenesis. Adeno-associated viral-mediated overexpression of Taz in osteoprogenitors alleviated bone loss and marrow fat accumulation in Bmncr-KO mice. Furthermore, restoring BMNCR levels in human BMSCs reversed the age-related switch between osteoblast and adipocyte differentiation. Our findings indicate that Bmncr is a key regulator of the age-related osteogenic niche alteration and cell fate switch of BMSCs.
MeSH Terms
- Adipocytes
- Adipogenesis
- Adiposity
- Aging
- Animals
- Bone Marrow
- Bone Morphogenetic Protein 2
- Fibromodulin
- Humans
- Mesenchymal Stem Cells
- Mice
- Mice, Knockout
- Osteoblasts
- Osteogenesis
- Osteoporosis
- RNA, Long Noncoding
- Signal Transduction
- Skeleton
Keywords
- Adult stem cells
- Endocrinology
- Noncoding RNAs
- Osteoporosis
Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.
MeSH Terms
- Alkaline Phosphatase
- Animals
- Antigens, CD
- Aorta
- Apyrase
- Bone Morphogenetic Protein 2
- Cell Proliferation
- Cells, Cultured
- Diphosphates
- Down-Regulation
- Durapatite
- Extracellular Space
- Gene Expression
- Hydrolysis
- Male
- Muscle, Smooth, Vascular
- Myocytes, Smooth Muscle
- Phosphates
- Phosphoric Diester Hydrolases
- Pyrophosphatases
- RNA, Messenger
- Rats, Sprague-Dawley
- Up-Regulation
- Vascular Calcification
Keywords
- aging
- alkaline phosphatase
- humans
- rats
- vascular calcification
Insulin-like growth factor binding proteins (IGFBP) play important roles in bone metabolism. IGFBP4 is involved in senescent-associated phenomena in mesenchymal stem cells (MSCs). The goal of the present study was to determine whether age-related IGFBP4 overexpression is associated with the impaired osteogenic differentiation potential of aged bone marrow derived MSCs. MSCs were isolated from Sprague-Dawley rats aged 3-26 months. The bone morphogenetic protein (BMP)-2-induced osteogenic differentiation of rat MSCs was assessed by analyzing the expression levels of osteoblast marker genes [runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OC)], ALP activity and calcification. Our study showed that IGFBP4 mRNA and protein expression increased with age in parallel with impaired osteogenic differentiation of MSCs cultured in BMP2-containing osteogenic medium, as evidenced by the downregulation of osteoblast marker genes, and decreased ALP activity and calcium deposits. IGFBP4 overexpression impaired BMP2-induced osteogenic differentiation potential of young MSCs, whereas IGFBP4 knockdown restored the osteogenic potency of aged MSCs. Moreover, IGFBP4 knockdown stimulated the activation of Erk and Smad by increasing phosphorylation. Collectively, our results demonstrate that IGFBP4 overexpression plays a role in the impairment of MSC differentiation potential via the Erk and Smad pathways, suggesting potential targets to improve MSC function for cell therapy applications.
MeSH Terms
- Animals
- Bone Marrow Cells
- Bone Morphogenetic Protein 2
- Cell Differentiation
- Cells, Cultured
- Gene Expression Regulation, Developmental
- Insulin-Like Growth Factor Binding Protein 4
- MAP Kinase Signaling System
- Mesenchymal Stem Cells
- Osteoblasts
- Osteogenesis
- Phosphorylation
- RNA, Messenger
- Rats
- Smad Proteins
Keywords
- Aging
- Bone marrow-derived mesenchymal stem cells
- Insulin-like growth factor binding protein-4
- Osteogenic differentiation
Bone mass is determined by the balance between bone formation, carried out by mesenchymal stem cell-derived osteoblasts, and bone resorption, carried out by monocyte-derived osteoclasts. Here we investigated the potential roles of p38 MAPKs, which are activated by growth factors and cytokines including RANKL and BMPs, in osteoclastogenesis and bone resorption by ablating p38α MAPK in LysM monocytes. p38α deficiency promoted monocyte proliferation but regulated monocyte osteoclastic differentiation in a cell-density dependent manner, with proliferating p38α cultures showing increased differentiation. While young mutant mice showed minor increase in bone mass, 6-month-old mutant mice developed osteoporosis, associated with an increase in osteoclastogenesis and bone resorption and an increase in the pool of monocytes. Moreover, monocyte-specific p38α ablation resulted in a decrease in bone formation and the number of bone marrow mesenchymal stem/stromal cells, likely due to decreased expression of PDGF-AA and BMP2. The expression of PDGF-AA and BMP2 was positively regulated by the p38 MAPK-Creb axis in osteoclasts, with the promoters of PDGF-AA and BMP2 having Creb binding sites. These findings uncovered the molecular mechanisms by which p38α MAPK regulates osteoclastogenesis and coordinates osteoclastogenesis and osteoblastogenesis.
MeSH Terms
- Aging
- Animals
- Bone Morphogenetic Protein 2
- Bone Remodeling
- Bone Resorption
- Cell Count
- Cell Differentiation
- Cell Proliferation
- Cyclic AMP Response Element-Binding Protein
- Integrases
- Male
- Mice
- Mitogen-Activated Protein Kinase 14
- Monocytes
- Osteoclasts
- Osteogenesis
- Osteoporosis
- Phenotype
- Platelet-Derived Growth Factor
- Stem Cells
- X-Ray Microtomography
The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.
MeSH Terms
- Absorptiometry, Photon
- Aging
- Animals
- Blotting, Western
- Disease Models, Animal
- Female
- Intercellular Signaling Peptides and Proteins
- Mice
- Mice, Inbred BALB C
- Microscopy, Electron, Scanning
- Osteogenesis
- Osteoporosis
- Peptides
- Vaccination
- Vaccines
- X-Ray Microtomography
Articular cartilage undergoes substantial age-related changes in molecular composition, matrix structure, and mechanical properties. These age-related differences between juvenile and adult cartilage manifest themselves as markedly distinct potentials for tissue repair and regeneration. To compare the biological properties and tissue regeneration capabilities of juvenile and adult bovine articular cartilage. Controlled laboratory study. Articular cartilage harvested from juvenile (age, 4 months) and adult (age, 6-8 years) bovine femoral condyles was cultured for 4 weeks to monitor chondrocyte migration, glycosaminoglycan content conservation, and new tissue formation. The cartilage cell density and proliferative activity were also compared. Additionally, the effects of age-related changes on cartilage gene expression were analyzed using the Affymetrix GeneChip array. Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly greater cell density, higher cell proliferation rate, increased cell outgrowth, elevated glycosaminoglycan content, and enhanced matrix metallopeptidase 2 activity. During 4 weeks in culture, only juvenile cartilage was able to generate new cartilaginous tissues, which exhibited pronounced labeling for proteoglycan and type II collagen but not type I collagen. With over 19,000 genes analyzed, distinctive gene expression profiles were identified. The genes mostly involved in cartilage growth and expansion, such as COL2A1, COL9A1, MMP2, MMP14, and TGFB3, were upregulated in juvenile cartilage, whereas the genes primarily responsible for structural integrity, such as COMP, FN1, TIMP2, TIMP3, and BMP2, were upregulated in adult cartilage. As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular, and molecular levels, the results strongly suggest that juvenile cartilage possesses superior chondrogenic activity and enhanced regenerative potential over its adult counterpart. Additionally, the differential gene expression profiles of juvenile and adult cartilage suggest possible mechanisms underlying cartilage age-related changes in their regeneration capabilities, structural components, and biological properties. The results of this comparative study between juvenile and adult bovine articular cartilage suggest an enhanced regenerative potential of juvenile cartilage tissue in the restoration of damaged articular cartilage.
MeSH Terms
- Aging
- Animals
- Cartilage, Articular
- Cattle
- Cell Count
- Cell Proliferation
- Chondrocytes
- Gene Expression Profiling
- Glycosaminoglycans
- Matrix Metalloproteinase 2
- Oligonucleotide Array Sequence Analysis
- Regeneration
Keywords
- adult
- aging
- articular cartilage
- biology of cartilage
- bovine
- cartilage regeneration
- cartilage repair
- chondrocyte
- gene expression
- juvenile
- knee
- migration
Cysteine (C)-X-C chemokine receptor-4 (CXCR4) is the primary transmembrane receptor for stromal cell-derived factor-1 (SDF-1). We previously reported in mouse or human bone marrow-derived mesenchymal stromal stem cells (BMSCs) that deleting or antagonizing CXCR4 inhibits bone morphogenetic protein-2 (BMP2)-induced osteogenic differentiation. The goal of this study was to determine whether CXCR4-deficiency in BMSCs is an age-related effect in association with impaired osteogenic differentiation potency of aged BMSCs. Using BMSCs derived from C57BL/6J wild type mice at ages ranging from 3 to 23 months old, we detected decreased CXCR4 mRNA and protein expression as well as SDF-1 secretion with advancing aging. Moreover, CXCR4-deficient BMSCs from elderly vs. young mice exhibited impaired osteogenic differentiation in response to BMP2 stimulation or when cultured in dexamethasone (Dex)-containing osteogenic medium, evidenced by decreased alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition (markers for immature and mature osteoblasts). Mechanistically, impaired BMP2- or Dex-osteoinduction in BMSCs of elderly mice was mediated by inhibited phosphorylation of intracellular R-Smads and Erk1/2 or Erk1/2 and p38 proteins, and decreased Runx2 and Osx expression (osteogenesis "master" regulators) were also detected. Furthermore, adenovirus-mediated repair of CXCR4 expression in BMSCs of elderly mice restored their osteogenic differentiation potentials to both BMP2 treatment and osteogenic medium. Collectively, our results demonstrate for the first time that CXCR4 expression in mouse BMSCs declines with aging, and this CXCR4-deficiency impairs osteogenic differentiation potency of aged BMSCs. These findings provide novel insights into mechanisms underlying age-related changes in BMSC-osteogenesis, and will potentiate CXCR4 as a therapeutic target to improve BMSC-based bone repair and regeneration in broad orthopedic situations.
MeSH Terms
- Adenoviridae
- Aging
- Animals
- Bone Marrow Cells
- Bone Morphogenetic Protein 2
- Cell Differentiation
- Culture Media
- Dexamethasone
- Enzyme Activation
- Extracellular Signal-Regulated MAP Kinases
- Humans
- Mesenchymal Stem Cells
- Mice
- Mice, Inbred C57BL
- Osteogenesis
- Receptors, CXCR4
- Smad Proteins
Keywords
- Aging
- Bone marrow mesenchymal stromal stem cells
- Bone morphogenetic protein-2
- CXC chemokine receptor-4
- Osteogenic differentiation