scholarly journals Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor.

1996 ◽  
Vol 133 (2) ◽  
pp. 457-468 ◽  
Author(s):  
C Shukunami ◽  
C Shigeno ◽  
T Atsumi ◽  
K Ishizeki ◽  
F Suzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Parathyroid Hormone ◽  
Chondrogenic Differentiation ◽  
Specific Binding ◽  
Receptor Gene ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Undifferentiated Cells ◽  
Cellular Condensation ◽  
Pthrp Receptor

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.

Down-regulation of chondrocyte aggrecan and type-II Collagen gene expression correlates with increases in static compression magnitude and duration

1999 ◽  
Vol 17 (6) ◽  
pp. 836-842 ◽  
Author(s):  
Paula M. Ragan ◽  
Alison M. Badger ◽  
Michael Cook ◽  
Vicki I. Chin ◽  
Maxine Gowen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type Ii Collagen ◽  
Collagen Gene ◽  
Type Ii ◽  
Down Regulation ◽  
Static Compression ◽  
Collagen Gene Expression

scholarly journals The transcription factors SP1 and MAZ regulate expression of the parathyroid hormone/parathyroid hormone-related peptide receptor gene

2000 ◽  
Vol 25 (3) ◽  
pp. 309-319 ◽  
Author(s):  
LJ Williams ◽  
AB Abou-Samra
Keyword(s):  
Transcription Factors ◽  
Parathyroid Hormone ◽  
Mutational Analysis ◽  
Receptor Gene ◽  
Dna Interaction ◽  
Osteosarcoma Cell ◽  
Specific Expression ◽  
Related Peptide ◽  
Its Gene ◽  
Pthrp Receptor

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor regulates extracellular calcium concentrations and is therefore important for mineral homeostasis. ROS 17/2.8 cells, a rat osteoblast-like osteosarcoma cell line, express the PTH/PTHrP receptor and provide a good model for examining the transcriptional regulation of its gene. The rat PTH/PTHrP receptor gene has two promoters, U1 and U3, which were shown to be important for its expression. Using extracts from ROS 17/2.8 cells, we have demonstrated two regions (termed FP1 and FP2) of nuclear protein/DNA interaction within promoter sequences previously shown to be important for the activity of the U3 promoter. Nuclear extracts from rat 2 fibroblasts, which do not express the PTH/PTHrP receptor, produced one site of protein/DNA interaction which was found at a position on the promoter identical to the position of FP1 produced by a ROS 17/2.8 nuclear extract. Mutation of these two sites of protein/DNA interaction resulted in reduced U3 promoter activity. Furthermore, we have demonstrated that the transcription factors SP1 and MAZ regulate U3 promoter expression and have shown their functional significance using mutational analysis. These data demonstrate that SP1 and MAZ bind to the PTH/PTHrP receptor promoter and that they are involved in cell-specific expression of its gene product.

scholarly journals Thecomparisonofserumbiomarkersin patients with osteoarthritisand rheumatoidarthritis

2018 ◽  
Vol 6 (2) ◽  
pp. 1-11
Author(s):  
Almandlawi S G ◽  
Ahmed A S
Keyword(s):  
Rheumatoid Arthritis ◽  
Vitamin D ◽  
Alkaline Phosphatase ◽  
Parathyroid Hormone ◽  
Calcium Phosphate ◽  
Serum Vitamin ◽  
Type Ii Collagen ◽  
Healthy Controls ◽  
Type Ii ◽  
Serum Vitamin D

Introduction: This study aims to assess the status of serum vitamin D, parathyroid hormone, type II collagen, calcium, phosphate,albumin, and alkaline phosphatase in osteoarthritis and rheumatoidarthritis patients and to study their association with rheumatoid arthritis disease activity. Materials and Methods: This prospectivecross-sectional study was conducted at the clinical analysis department, College of Pharmacy, Hawler Medical University in 2017.They study samples were collected at Rizgary Teaching Hospitalduring the period September 2015 to January 2016. A total of(N=156) participants were included: (N=53) patients with rheumatoid arthritis (RA), (N=53) with osteoarthritis (OA), and (N=50)healthy controls. Enzyme Linked Immuno Sorbent Assay kits determined serum vitamin D, parathyroid hormone, and type II collagen; and serum albumin, calcium, phosphate and alkaline phosphatase, were determined by standard colorimetric methods. Resultsand Discussion: Statistically significant higher levels of parathyroid hormone and type II collagen, with lower levels of Vitamin D,were found in the osteoarthritis group than the rheumatoid arthritisgroup and the healthy controls (P=0.007, P<0.001, P= 0.005) respectively. Multiple linear regression showed a statistically significant difference in serum type II collagen as a dependent variable, inpatients suffering from RA or OA compared to the healthy controlgroup; after adjusting for the effect of other independent studyvariables, there was a mean increase of (45.90 nmol/L, P<0.001)in RA patients, and OA patients showed greater levels of type IIcollagen (73.950 nmol/L) than the health control group (P<0.001).Conclusions: Elevated type II collagen levels, in conjunction witha low vitamin D status, may be strong discriminator between osteoarthritis and rheumatoid arthritis patients.

scholarly journals A Novel Van91 I Polymorphism in the 1st Intron of the Parathyroid Hormone(PTH)/PTH-Related Peptide(PTHrP) Receptor Gene and Its Effect on the Urinary cAMP Response to PTH.

2000 ◽  
Vol 23 (4) ◽  
pp. 386-389 ◽  
Author(s):  
Masayuki HEISHI ◽  
Hiromitsu TAZAWA ◽  
Takatoshi MATSUO ◽  
Takayuki SARUTA ◽  
Masato HANAOKA ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Receptor Gene ◽  
Related Peptide ◽  
Pthrp Receptor

scholarly journals FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor β1 signaling

2019 ◽  
Vol 294 (46) ◽  
pp. 17555-17569 ◽  
Author(s):  
Ichiro Kurakazu ◽  
Yukio Akasaki ◽  
Mitsumasa Hayashida ◽  
Hidetoshi Tsushima ◽  
Norio Goto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cell Cycle Arrest ◽  
Chondrogenic Differentiation ◽  
Transforming Growth Factor ◽  
Human Mesenchymal Stem Cells ◽  
Type Ii Collagen ◽  
Transforming Growth Factor Β1 ◽  
Type Ii ◽  
Cycle Arrest

The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFβ1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFβ1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9. To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFβ1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFβ1 signaling.

scholarly journals Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study

2020 ◽  
Vol 2020 ◽  
pp. 1-16 ◽  
Author(s):  
Christel Henrionnet ◽  
Léa Pourchet ◽  
Paul Neybecker ◽  
Océane Messaoudi ◽  
Pierre Gillet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Cell Encapsulation ◽  
Type Ii Collagen ◽  
Mitochondrial Activity ◽  
Type Ii ◽  
3D Bioprinting ◽  
Matrix Synthesis ◽  
M Cell ◽  
Low Concentration

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10×10×4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

Effects of bio-mimic collagen II-g-hyaluronic acid copolymers on chondrocyte maintenance

2019 ◽  
Vol 34 (4-5) ◽  
pp. 373-385
Author(s):  
Kuan Wei Lee ◽  
Tang-Ching Kuan ◽  
Ming Wei Lee ◽  
Chen Show Yang ◽  
Lain-Chyr Hwang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Hyaluronic Acid ◽  
Specific Binding ◽  
Type Ii Collagen ◽  
Polymerase Chain Reaction Analysis ◽  
Collagen Fibrils ◽  
Type Ii ◽  
Human Cartilage

Extracellular matrix has an important part of the role in tissue engineering and regenerative medicine, so it is necessary to understand the various interactions between cells and extracellular matrix. Type II collagen and hyaluronic acid are the major structural components of the extracellular matrix of articular cartilage, and they are involved in fibril formation, entanglement and binding. The aim of this study was to prepare type II collagen fibrils with surface grafted with hyaluronic acid modified at the reducing end. The topographic pattern of type II collagen fibrils showed a significant change after the surface coupling of hyaluronic acid according to atomic force microscopy scanning. The presence of hyaluronic acid on the type II collagen fibrillar surface was confirmed by the specific binding of nanogold labelled with lectin. No significant increase in cell proliferation was detected by a WST-1 assay. According to histochemical examination, the maintenance of the round shape of chondrocytes and increased glycosaminoglycan secretion revealed that these cell pellets with Col II- g-hyaluronic acid molecules contained un-dedifferentiated chondrocytes in vitro. In the mixture with the 220-kDa Col II- g-hyaluronic acid copolymer, the expression of type II collagen and aggrecan genes in chondrocytes increased as demonstrated by real-time polymerase chain reaction analysis. Experimental results show that the amount of hyaluronic acid added during culturing of chondrocytes can maintain the functionality of chondrocytes and thus allow for increased cell proliferation that is suitable for tissue repair of human cartilage.

scholarly journals Demonstration of immunoreactive sites on cartilage after in vivo administration of biotinylated anti-type II collagen antibodies.

1989 ◽  
Vol 37 (2) ◽  
pp. 265-268 ◽  
Author(s):  
R Jonsson ◽  
A L Karlsson ◽  
R Holmdahl
Keyword(s):  
Monoclonal Antibodies ◽  
Simple Technique ◽  
Specific Binding ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Cytochemical Staining ◽  
Collagen Antibodies ◽  
Detection And Localization ◽  
In Vivo Administration

Administration of biotinylated monoclonal antibodies provides the basis of a simple technique for identifying immunoreactive sites in vivo. Biotinylated anti-type II collagen antibodies were injected intraperitoneally into normal DBA/1 mice. The mice were sacrificed after 96 hr and the front paws removed and decalcified to allow tissue sectioning before snap-freezing. Binding of antibodies in vivo was visualized with affinity cytochemical staining using avidin-biotin-peroxidase complexes. Specific binding of antibodies to cartilaginous structures was seen after injection of 20-500 micrograms biotinylated monoclonal or polyclonal anti-type II collagen antibodies, but not after injection of a biotinylated control antibody. This technique should further the detection and localization studies of tissue components involved in the dynamics of physiological and pathological processes.

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