Osteoblast iron genes: real time PCR and microarray hybridization approach under hyperoxia

2021 ◽  
Vol 32 (4) ◽  
pp. 491-496
Author(s):  
Prihartini Widiyanti ◽  
Hartmut Kuehn ◽  
Soetjipto Soetjipto

Abstract Objectives Iron is essential for cell growth, differentiation, electron transfer, and oxygen transport. Hyperoxia may increase the turnover of bone matrix components with a net effect of accelerated bone growth. Although hyperoxia was claimed could increase osteoblast activity, but expression level in possible genes which play role in proliferation is still unclear. This research aims to prove the differences of expression level of transferrin receptor gene and iron regulated transporter and other genes of 7F2 under 24 h normoxia, 24 h hyperoxia, and 48 h hyperoxia and the effect of hyperoxia by using osteoblast cell culture 7F2. Methods Reverse transcriptase, real time Polymerase Chain Reaction (PCR), and microarray is used to qualitatively detect gene expression. The computer softwares such as National Center for Biotechnology Information (NCBI) data base, Software Affymetrix, DNA Strider program, Genomatix – DiAlign program, Oligo 5.0 program (Software primer design) from Wojciech & Piotr Rychlik, and Genetyx-Mac version 8.0 have been used to analyze the PCR result. Results Under 24 h hyperoxia, there were 3,884 copies of transferrin receptor mRNA per 1,000,000 copies of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. After 24 h hyperoxia, 8,325 copies of transferrin receptor mRNA per 1,000,000 GAPDH mRNA copies were found showing 2.1-fold up regulation. After 48 h hyperoxia, there was no significant increase at the level of expression of transferrin receptor mRNA, 8,079 mRNA copies per 1,000,000 copies of mRNA were found (2.0-fold up regulation compared with 24 h normoxia). Conclusions It can be concluded that hyperoxia might have an effect on upregulating the expression of some osteoblast genes which might have an impact on osteoblast activity.

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3352-3352
Author(s):  
Daniel Garcia dos Santos ◽  
Matthias Schranzhofer ◽  
Nam Lok Chun ◽  
Amel Hamdi ◽  
Prem Ponka

Abstract The transferrin receptor (TfR) is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron (Fe) from a plasma glycoprotein, transferrin. Iron uptake from diferric transferrin (Tf) involves the binding of transferrin to the TfR followed by internalization of Tf within an endocytic vesicle by receptor-mediated endocytosis. Iron is then released from transferrin within endosomes by a combination of Fe3+ reduction by Steap3 (likely when transferrin is still bound to TfR) and a decrease in pH (~pH 5.5). Following this, Fe2+ is transported across the endosomal membrane by DMT1. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of TfR is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3' untranslated region (UTR) of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (iron regulatory proteins; IRPs) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected, since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. We previously documented that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis (1). In this study we have demonstrated that incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is primarily a result of increased mRNA synthesis, since the effect of ALA can be abolished by actinomycin D. Moreover, we found that the TfR promoter was activated in vitro by the addition of ALA or hemin to murine erythroleukemia (MEL) cells induced to differentiate using DMSO. Furtehermore, site-directed mutation of erythroid active element (1) in the TfR promoter abolished the effects of ALA or hemin. These results indicate that heme may directly or indirectly interact with the TfR promoter, consequently enhancing the gene expression. Hence, our results show that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. In conclusion, erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels. 1 Chun-Nam Lok Ponka P. (2000) Identification of an Erythroid Active Element in the Transferrin Receptor Gene. J. Biol. Chem. 275: 24185-24190. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Qiufang Wang ◽  
Yumei Shen

This paper combines flexible pressure sensing technology, wireless sensor network, and cloud platform technology to design and manufacture a medical miniature pressure sensor and its supporting system. The problem of noninvasive monitoring of the syndrome encountered in the clinic is used for real-time monitoring and auxiliary diagnosis of the disease. Different from the current clinical use of “puncture” to measure intrafascial pressure, this system focuses on the noninvasive monitoring of compartment syndrome, using medical tape to paste a flexible microsensing unit on the injured area. The flexible sensor unit can measure the pressure here in real time and then can know the pressure in the fascia chamber. The flexible pressure sensor unit combines with the subsequent flexible circuit to send the measured data to the data in real time through wireless communication. The data aggregation node transmits the collected data to the upper computer through serial communication, and the upper computer software processes and stores the data and uploads it to the cloud server. In this experiment, it was observed that the concentrations of Ca and P showed the same fluctuating trend. With the gradual progress of the stretch, the concentrations of Ca and P increased with the increase in time, reaching approximately at the end of the extension. The peak value indicates that the osteoclast activity is enhanced at this time, the bone matrix is largely destroyed, and the Ca and P in the matrix are released into the serum in a large amount, thereby increasing the serum concentration. After the distraction ceases, it enters the healing period of the callus. At this time, the concentrations of Ca and P decrease with the increase in time and gradually reach a stable level, indicating that the osteoblast activity is enhanced at this time, the bone matrix begins to rebuild, and the Ca and P gradually increase. The deposited bone matrix gradually forms new bone and finally reaches a balance. Since the speed of extension in each experimental group is inconsistent, the time required to reach the same extension length is also inconsistent, so that the peak time is also inconsistent. After plotting the stress difference ( △ F ) before and after stretching against time and speed, it is found that the relationship is linear. However, these two variables affect △ F at the same time, so they cannot be isolated. Based on this, this subject uses multiple regression equations to fit the three relationships of stress difference ( △ F ), time, and speed. In the process of distraction osteogenesis, with each distraction, the bone stress presents a trend from high to low. And as the stretch progresses, the measured stress value increases linearly at the same time point every day.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 665-665 ◽  
Author(s):  
Yoshio Katayama ◽  
Andres Hidalgo ◽  
Paul S. Frenette

Abstract The exact mechanisms mediating G-CSF-induced hematopoietic progenitor cell (HPC) egress from the bone marrow (BM) are incompletely understood. Recent studies have suggested that the degradation of SDF-1 in the BM by G-CSF-induced proteolysis may play an important role. We previously hypothesized that endogenous galactocerebrosides (GCs) might be involved in HPC trafficking since certain sulfogalactolipids share biological properties with fucoidan, a sulfated fucose polymer endowed with mobilization activity, and showed that G-CSF fails to induce HPC mobilization in mice lacking UDP-galactose:ceramide galactosyl transferase (CGT) (Blood 2001 98:811a), the enzyme necessary for GC synthesis. To gain further mechanistic insights, we assessed protease activity and found no difference in elastase release from BM cells and in the degradation of exogenous SDF-1 in BM extracellular fluid (BMEF) between CGT−/− and +/+ littermates. Furthermore, endogenous SDF-1 levels in BMEF of CGT−/− and +/+ mice showed a similar reduction after G-CSF stimulation (>50% in CGT−/− mice, n=7–9, p<0.05) despite a virtual absence of mobilization. These data suggest that the reduction of SDF-1 in bone marrow is not essential for G-CSF-induced mobilization. To evaluate the spacial distribution of SDF-1 expression in mouse BM, we stained SDF-1 using the tyramide amplification system. We found that SDF-1 staining was sparsely distributed in the BM but, surprisingly, strong homogenous staining was observed in the surrounding bone. Staining specificity was confirmed by ELISA (2.6±0.5 vs 5.8±1.0 ng SDF-1 per femur for BMEF and bone protein extracts, respectively, n=8, p<0.05). Following G-CSF stimulation, SDF-1 protein levels were significantly decreased in bone extracts from CGT+/+ littermates (53% reduction, n=4–5, p<0.05), but were virtually unchanged in CGT−/− mice. Quantitative real-time RT-PCR analyses revealed that SDF-1 was transcriptionally downregulated by G-CSF in both BM and bone in CGT+/+ mice but there was no significant reduction in CGT−/− bone. Since osteoblasts may represent a major source of SDF-1, we suspected that osteoblast activity might be altered in CGT−/− mice. We thus measured plasma osteocalcin levels by ELISA and found a significant reduction in CGT−/− mice compared to CGT+/+ littermates (39% reduction, n=6–9, p<0.001). Immunofluorescence experiments revealed that bone lining osteoblasts in CGT−/− mice were flattened and small while in CGT+/+ littermates, these cells displayed a healthy cobblestone-like appearance. Furthermore, there was a trend toward reduction of gene expression in Runx2, a critical transcription factor in osteoblasts, and α1(I) collagen, an osteoblast-specific bone matrix protein, in CGT−/− BM by real-time RT-PCR. These data suggest that dysregulation of bone SDF-1 in CGT−/− mice may be due to constitutive downregulation of osteoblastic activity. Strikingly, Runx2 and α1(I) collagen were dramatically downregulated by G-CSF in CGT+/+ BM (Runx2; 65% reduction, n=4, p<0.001, α1(I) collagen; 92% reduction, n=4, p<0.05 by real-time RT-PCR). G-CSF does not appear to act directly on osteoblasts since G-CSF receptor mRNA was not detectable in primary osteoblast and 4 different osteoblast lineage cell lines. In conclusion, bone SDF-1, rather than that of BM, may regulate HPC mobilization. The abnormal regulation of bone SDF-1 and reduced osteoblastic activity in CGT−/− mice strongly suggest that bone SDF-1 originates from osteoblasts and that a rapid downregulation of osteoblastic activity may play a key role in the egress of stem cells from BM.


Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 12
Author(s):  
David Chavarri-Prado ◽  
Aritza Brizuela-Velasco ◽  
Ángel Álvarez-Arenal ◽  
Markel Dieguez-Pereira ◽  
Esteban Pérez-Pevida ◽  
...  

Objectives: To determine the effect of mechanical loading of bone on the stability and histomorphometric variables of the osseointegration of dental implants using an experimental test in an animal model. Materials and Methods: A total of 4 human implants were placed in both tibiae of 10 New Zealand rabbits (n = 40). A 6-week osseointegration was considered, and the rabbits were randomly assigned to two groups: Group A (Test group) included 5 rabbits that ran on a treadmill for 20 min daily during the osseointegration period; Group B (Controls) included the other 5 that were housed conventionally. The monitored variables were related to the primary and secondary stability of the dental implants (implant stability quotient—ISQ), vertical bone growth, bone to implant contact (BIC), area of regenerated bone and the percentage of immature matrix. Results: The results of the study show a greater vertical bone growth (Group A 1.26 ± 0.48 mm, Group B 0.32 ± 0.47 mm, p < 0.001), higher ISQ values (Group A 11.25 ± 6.10 ISQ, 15.73%; Group B 5.80 ± 5.97 ISQ, 7.99%, p = 0.006) and a higher BIC (Group A 19.37%, Group B 23.60%, p = 0.0058) for implants in the test group, with statistically significant differences. A higher percentage of immature bone matrix was observed for implants in the control group (20.68 ± 9.53) than those in the test group (15.38 ± 8.84) (p = 0.108). A larger area of regenerated bone was also observed for the test implants (Group A 280.50 ± 125.40 mm2, Group B 228.00 ± 141.40 mm2), but it was not statistically significant (p = 0.121). Conclusions: The mechanical loading of bone improves the stability and the histomorphometric variables of the osseointegration of dental implants.


2010 ◽  
Vol 27 ◽  
pp. S66
Author(s):  
M. Piechota ◽  
A. Banaszewska ◽  
E. Guzniczak ◽  
G. Rosinski ◽  
T. Siminiak ◽  
...  

1992 ◽  
Vol 135 (3) ◽  
pp. 459-468 ◽  
Author(s):  
K. L. Hull ◽  
R. A. Fraser ◽  
S. Harvey

ABSTRACT Although GH has no direct effect on GH release from chicken pituitary glands, GH receptor mRNA similar to that in the rabbit liver was identified by Northern blot analysis in extracts of adult chicken pituitaries. Complementary (c) DNA, reverse transcribed from chicken pituitary RNA, was amplified by the polymerase chain reaction (PCR) in the presence of 3′- and 5′-oligonucleotide primers coding for the extracellular domain of the chicken liver GH receptor and was found to contain an electrophoretically separable fragment of 500 bp, identical in size to that in chicken liver. Digestion of this pituitary cDNA with NcoI produced expected moities of 350 and 150 bp. Amplification of chicken pituitary cDNA in the presence of oligonucleotide primers for the intracellular sequence of the chicken liver GH receptor produced an electrophoretically separable fragment of approximately 800 bp, similar to that in chicken liver. This fragment was cut into expected moieties of 530 and 275 bp after digestion with EcoRI. These PCR fragments were identified in extracts of the pituitary caudal lobe, in which somatotrophs are confined and account for the majority of endocrine cell types, and in the cephalic lobe, in which somatotrophs are lacking. Translation of the GH receptor mRNA in the pituitary gland was indicated by the qualitative demonstration of radio-labelled GH-binding sites in plasma membrane preparations, in pituitary cytosol and in nuclear membranes. These results provide evidence for the expression and translation of the GH receptor gene in pituitary tissue, in which GH receptors appear to be widely distributed within cells and in different cell types. GH may therefore have paracrine, autocrine or intracrine effects on pituitary function. Journal of Endocrinology (1992) 135, 459–468


Endocrinology ◽  
1999 ◽  
Vol 140 (5) ◽  
pp. 2110-2116 ◽  
Author(s):  
Roni Mamluk ◽  
Nitzan Levy ◽  
Bo Rueda ◽  
John S. Davis ◽  
Rina Meidan

Abstract Our previous studies demonstrated that endothelin-1 (ET-1), a 21-amino acid vasoconstrictor peptide, has a paracrine regulatory role in bovine corpus luteum (CL). The peptide is produced within the gland where it inhibits progesterone production by acting via the selective type A endothelin (ETA) receptors. The present study was designed to characterize ETA receptor gene expression in different ovarian cell types and its hormonal regulation. ETA receptor messenger RNA (mRNA) levels were high in follicular cells as well as in CL during luteal regression. At this latter stage, high ETA receptor expression concurred with low prostaglandin F2α receptor mRNA. The ETA receptor gene was expressed by all three major cell populations of the bovine CL; i.e. small and large luteal cells, as well as in luteal endothelial cells. Among these various cell populations, the highest ETA receptor mRNA levels were found in endothelial cells. cAMP elevating agents, forskolin and LH, suppressed ETA receptor mRNA expression in luteinized theca cells (LTC). This inhibition was dose dependent and was evident already after 24 h of incubation. In luteinized granulosa cells (LGC), 10 and 100 ng/ml of insulin-like growth factor I and insulin (only at a concentration of 2000 ng/ml) markedly decreased ETA receptor mRNA levels. In both LGC and LTC there was an inverse relationship between ETA receptor gene expression and progesterone production; insulin (in LGC) and forskolin (in LTC) enhanced progesterone production while inhibiting ETA receptor mRNA levels. Our findings may therefore suggest that, during early stages of luteinization when peak levels of both LH and insulin-like growth factor I exist, the expression of ETA receptors in the gland are suppressed. This study demonstrates physiologically relevant regulatory mechanisms controlling ETA receptor gene expression and further supports the inhibitory role of ET-1 in CL function.


1986 ◽  
Vol 6 (12) ◽  
pp. 4244-4250
Author(s):  
L M Neckers ◽  
S Bauer ◽  
R C McGlennen ◽  
J B Trepel ◽  
K Rao ◽  
...  

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.


1988 ◽  
Vol 526 (1 Hemochromatos) ◽  
pp. 54-64 ◽  
Author(s):  
John L. Casey ◽  
Bruno Jeso ◽  
Krishnamurthy Rao ◽  
Tracey A. Rouault ◽  
Richard D. Klausner ◽  
...  

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