Introduction
Osteoblasts are bone-forming cells differentiated from mesenchymal stem cells (MSCs) that reside in the bone marrow. The differentiation of MSCs into osteoblasts is elaborately regulated by essential transcription factors including runt-related transcription factor 2 (Runx2), osterix, and distal-less homeobox 5 (Dlx5) [11]. Upon differentiation, osteoblasts deposit and mineralize the bone matrix by secreting proteoglycans, glycoproteins, and γ-carboxylated (Gla) proteins, which are crucial for regulating adhesion, migration, proliferation, and differentiation of bone cells [3]. Osteoblasts are required for maintaining bone homeostasis [28] and use glucose as their primary energy source. Glucose is transported into cells through glucose transporter 1 (Glut1) and Glut3, depending on the intracellular glucose concentration [29, 32]. Although high glucose levels do not alter Glut1 expression in osteoblasts [4], they induce bone loss by decreasing the function of osteoblasts [34].
Hyperglycemia is characterized by high blood glucose levels and diverse physiological effects [2, 8, 19]. It increases the production of reactive oxygen species (ROS) and disrupts mitochondrial function [10]. In particular, in osteoblasts, it inhibits cell proliferation [9], enhances cell apoptosis [1], and decreases the expression of Runx2, Dlx5, and bone morphogenetic proteins, leading to reduced osteoblast differentiation and an increased number of immature osteoblasts [4]. Consequently, there is a gradual loss in the mineralization function of osteoblasts, causing bone loss. In addition, hyperglycemia-induced ROS production in osteoblasts affects mitochondrial function by disrupting the balance between mitochondrial fission and fusion [9, 10].
Unique cartilage matrix-associated protein (UCMA) was first discovered in cartilage and identified as one of the vitamin K-dependent proteins (VKDPs) [24, 26]. UCMA is γ-carboxylated by vitamin K-dependent γ-glutamyl carboxylase, resulting in an increased calcium-binding affinity [7]. It accelerates osteoblast differentiation and calcium deposition in the bone [14] and inhibits ectopic calcification in the vascular system [33]. Other extrahepatic VKDPs, matrix Gla protein (MGP) and osteocalcin (OC), also play important roles in calcification [24]. It has been reported that OC inhibits ROS production induced by high glucose in osteoblasts [18]. However, the potential effect of UCMA under high glucose conditions has not yet been elucidated. Therefore, we investigated the alleviating effect of UCMA on hyperglycemic stress regulation in MC3T3-E1 osteoblasts.
Materials and Methods
Cell culture
MC3T3-E1 osteoblastic cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in α-minimum essential medium (HyClone, Logan, UT, USA) containing the 5.5 mM glucose concentration, which was supplemented with 10% fetal bovine serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, CA, USA) at 37℃ in an atmosphere of 5% CO2. Next, the cells were seeded at a density of 2×103 cells/well in 8-chamber slides for ROS estimation and at a density of 3×104 cells/well in 6-well plates for RNA and protein extraction. Then, the cells were treated with 30.5 mM D-(+)-glucose (Sigma-Aldrich, St. Louis, MO, USA) with or without 5 μg/ml UCMA-FLAG protein for 24 or 72 hr, which were designated as high glucose and UCMA, respectively. The extraction and purification of UCMA-FLAG protein were performed by Thermo Fisher Scientific (Waltham, MA, USA) using the following method. Briefly, the expression vector containing the gene encoding UCMA-FLAG was prepared as previously described [14]. The vector was transfected into Expi293 cells, and the cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 0.1% Triton X-100, followed by sonication. The culture supernatants with the secreted UCMA-FLAG protein were centrifuged at 14,000 rpm for 1 min, and the UCMA-FLAG protein was purified through the FLAG-tag affinity column.
Determination of ROS production
MC3T3-E1 cells were treated with high glucose and UCMA for 24 hr and incubated in a medium containing 5 μM CellROX Green (Invitrogen, Waltham, MA, USA) for 30 min or 5 μM MitoSOX Red (Invitrogen) for 15 min in the dark. The cells were then mounted in an antifade solution containing 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). CellROX- and MitoSOX-positive cells were detected at 485/520 and 510/580 nm, respectively, using a fluorescence microscope (Nuance FX). The fluorescence intensity was quantified using the ImageJ software (NIH, Bethesda, MD, USA).
Immunocytochemistry
After treating the MC3T3-E1 cells with high glucose and UCMA for 24 hr, they were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.01% Triton X-100 for 10 min, and blocked with 10% bovine serum albumin (Sigma-Aldrich) for 30 min at room temperature. After blocking, the cells were incubated overnight at 4℃ with an anti-heme oxygenase 1 (HO-1) antibody (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation for 1 hr with Alexa Fluor 488-conjugated secondary antibody (Invitrogen). Then, the cells were mounted in an antifade solution containing DAPI to counterstain the nuclei. All images were obtained using a fluorescence microscope (Leica, Wetzlar, Germany).
RNA extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from the cells cultured for 72 hr using TRIzol reagent (Sigma-Aldrich), according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcriptase premix (ELPIS-Biotech, Daejeon, Korea) with 1 µg of total RNA, followed by qPCR using Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) on a StepOnePlus real-time PCR system (Applied Biosystems). The cycle progressed as follows: denaturation at 94℃ for 2 min; 40 cycles of amplification with denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s; and a final extension at 72℃for 10 min. The following primers were used for qPCR: nuclear factor erythroid 2-related factor 2 (Nrf2, 5'-CTT AGA GGC TCA TCT CAC AC-3' and 5'-CAG CTT CCT TTT CCT ACA GT-3'), superoxide dismutase 1 (SOD1, 5'-CCA TCA GTA TGG GGA CAA TAC A-3' and 5'-GGT CTC CAA CAT GCC TCT CT-3'), SOD2 (5'-GAC CCA TTG CAA GGA ACA A-3' and 5'-GTA GTA AGC GTG CTC CCA CAC-3'), and glyceraldehyde 3-phosphate dehydrogenase (Gapdh, 5'-CAT CTC CCT CAC AAT TTC CA-3' and 5'-GTG CAG CGA ACT TTA TTG ATG G-3'). Results were analyzed using the comparative cycle threshold (CT) method.
Protein extraction and western blotting
Total protein was extracted from the cultured cells using radioimmunoprecipitation assay buffer (Elpis Biotech) containing proteinase K (Roche, Basel, Switzerland). Equal amounts of protein were separated by 4%-20% mini-protein TGX precast protein gel (Bio-Rad, Hercules, CA, USA) electrophoresis and transferred onto PVDF membranes (Millipore, Burlington, MA, USA) according to standard immunoblot procedures. The membranes were incubated overnight at 4℃ with the following antibodies: anti-dynamin-related 1 (DRP1; Cell Signaling Technology, Danvers, MA, USA, 8570S, 1:1,000), anti-mitofusin-2 (MFN2; Cell Signaling Technology, 9482S, 1:1,000), anti-AKT (Cell Signaling Technology, #4685, 1:1,000), anti-phospho-AKT (p-AKT; Cell Signaling Technology, #4058, 1:1,000), and anti-β-ACTIN (Santa Cruz Biotechnology, sc-47778, 1:1,000). Bound anti-bodies were detected using an enhanced chemiluminescence kit (Bio-Rad). The intensities of protein bands were analyzed using the ImageJ software.
Statistical analysis
All data were analyzed using the Student’s t-test and were presented as mean ± standard deviation (SD). A p value <0.05 was considered statistically significant.
Results
UCMA inhibited high glucose-induced ROS production in osteoblasts
Previous studies have demonstrated that UCMA treatment promotes the differentiation and nodule formation in MC3T3-E1 cells, suggesting its promising role in osteoblasts [14]. Another VKDP, OC, not only plays a critical role in calcification but also inhibits ROS production at high glucose levels [18, 24]. Based on this information, we investigated whether UCMA affects high glucose-induced ROS production in MC3T3-E1 cells treated with high concentrations of glucose, with or without UCMA. CellROX and MitoSOX staining were performed to determine cellular and mitochondrial ROS production, respectively. The results of CellROX and MitoSOX staining revealed increased ROS production in the cytoplasm and mitochondria of high glucose-treated MC3T3-E1 cells, respectively, which subsequently decreased upon the addition of UCMA (Fig. 1A). Moreover, quantification of CellROXand MitoSOX-stained cells showed the increased intracellular ROS production in the cytoplasm and mitochondria of high glucose-treated cells, which decreased after subsequent treatment with UCMA (Fig. 1B).
Fig. 1. ROS production in UCMA- and high glucose-treated osteoblasts. MC3T3-E1 cells were treated with NG, HG, or HG + UCMA for 24 hr. (A) CellROX and MitoSOX staining were performed to assess intracellular and mitochondrial membrane ROS production, respectively. Green and red indicate ROS stained by CellROX and MitoSOX, respectively; blue indicates the nucleus stained by DAPI. Scale bar, 5 μm. (B) The relative fluorescence intensity is plotted against the fluorescence intensity of NG-treated cells. Quantitative data are presented as mean ± SD. *p<0.05 compared to NG, #p<0.05 compared to HG. CellROX (n=5), MitoSOX (n=3). ROS, reactive oxygen species; UCMA, unique cartilage matrix-associated protein; NG, normal glucose; HG, high glucose; DAPI, 4',6-diamidino-2-phenylindole.
To confirm the inhibition of ROS production by UCMA, the mRNA expression of antioxidant genes was measured using qPCR. The mRNA expression of Nrf2 and SOD1 tended to decrease under high glucose conditions compared with that under normal glucose conditions in MC3T3-E1 cells, and the levels were significantly reversed by UCMA addition (Fig. 2A). SOD2 expression, however, showed no significant differences among the three groups (Fig. 2A). Furthermore, exposure to high glucose concentrations resulted in HO-1 translocation from the cytoplasm to nucleus (Fig. 2B). Interestingly, UCMA treatment reduced the increase in nuclear HO-1 expression induced by high glucose concentrations (Fig. 2B). Collectively, these results suggested that UCMA ameliorated the high glucose-induced ROS production in osteoblasts.
Fig. 2. Expression of antioxidant genes and HO-1 in UCMA- and high glucose-treated osteoblasts. (A) The mRNA expression of antioxidant genes was analyzed using qPCR. RNA was extracted from MC3T3-E1 cells treated with NG, HG, and HG + UCMA for 72 hr. The mRNA levels of Nrf2, SOD1, and SOD2 were normalized to the expression level of Gapdh, and the relative mRNA expression levels were plotted against gene expression levels in NG-treated cells. Quantitative data are presented as mean ± SD. #p<0.05 compared to HG. NG (n=5), HG (n=5), HG + UCMA (n=4). (B) Immunocytochemistry was performed to detect the expression of HO-1. MC3T3-E1 cells were treated with NG, HG, and HG + UCMA for 24 hr. Green indicates HO-1 expression; blue indicates the nucleus stained by DAPI. Scale bar, 5 μm. UCMA, unique cartilage matrix-associated protein; NG, normal glucose; HG, high glucose; Nrf2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; HO-1, heme oxygenase 1; DAPI, 4',6-diamidino-2-phenylindole.
UCMA increased mitochondrial fission in response to high glucose-induced ROS production
High glucose concentrations cause dysregulation of mitochondrial fission and fusion processes leading to ROS generation [36, 37]. DRP1 and MFN2 are involved in mitochondrial fission and fusion, respectively [10]. To investigate dysregulated mitochondrial fission and fusion, western blotting was performed using anti-DRP1 and anti-MFN2 antibodies. Although DRP1 expression did not differ between cells treated with high glucose alone and cells treated with normal glucose, its expression was increased in cells treated with both high glucose and UCMA compared with that in cells treated with high glucose alone (Fig. 3A). However, MFN2 expression showed no significant changes in any of the treatment groups (Fig. 3B). These results suggested that UCMA regulates mitochondrial fission by decreasing the high glucose-induced ROS production.
Fig. 3. Altered mitochondrial fission in UCMA-treated osteoblasts. Western blotting was performed to confirm the expression of proteins associated with mitochondrial fission using an anti-DRP1 antibody (A) and with mitochondrial fusion using an anti-MFN2 antibody (B). Proteins were extracted from MC3T3-E1 cells treated with NG, HG, and HG + UCMA for 72 hr. Quantitative analyses were performed, and the data are presented as mean ± SD. #p<0.05 compared to HG (n=3). Numbers represent experiments that were independently performed and collected in triplicate. UCMA, unique cartilage matrix-associated protein; NG, normal glucose; HG, high glucose; DRP1; dynamin-related protein 1; MFN2, mitofusin-2.
UCMA altered AKT signaling in response to high glucose-induced ROS production
AKT signaling is primarily involved in high glucose-induced ROS production [38]. To investigate the activation of AKT signaling, we performed western blotting using anti-AKT and anti-p-AKT antibodies to determine total AKT and activated AKT, respectively. The total expression of AKT showed no difference in any of the treatment groups. Consistent with DRP1 expression, the expression ratio of p-AKT/total AKT remained unchanged in cells treated with high glucose only compared with that in cells treated with normal glucose. However, the expression ratio of p-AKT/total AKT was significantly reduced in cells treated with both high glucose and UCMA compared with that in cells treated with high glucose only (Fig. 4). These results suggested that UCMA inhibits AKT signaling by decreasing the high glucose-induced ROS production.
Fig. 4. Altered AKT signaling in UCMA-treated osteoblasts. Western blotting was performed to confirm the activation of AKT using anti-AKT and anti-p-AKT antibodies. Proteins were extracted from MC3T3-E1 cells treated with NG, HG, and HG + UCMA for 72 hr. Quantitative analyses were performed, and the data are presented as mean ± SD. #p<0.05 compared to HG (n=3). Numbers represent experiments that were independently performed and collected in triplicate. UCMA, unique cartilage matrix-associated protein; NG, normal glucose; HG, high glucose; p-AKT, phospho-AKT.
Discussion
UCMA is a secretory protein that belongs to the VKDP family and is primarily detected in cartilaginous tissues, bones, skin, and the vascular system [26, 30, 31]. Due to its Gla-rich composition, UCMA has a high binding affinity for calcium and thus plays a role in calcium deposition in bones [25]. Nonetheless, other functions of UCMA have also been demonstrated. Reportedly, UCMA inhibits cartilage degradation by regulating aggrecanase activity in inflammatory arthritis [23] and promotes osteoblast differentiation by directly binding to fibrillin-2 in osteoblasts [15]. It also exhibits anticancer properties by inhibiting the migration and invasion of triple-negative breast cancer cells [13]. Among VKDPs, OC ameliorates high glucose-induced ROS production through the AKT signaling pathway in osteoblasts [18]. Moreover, an increase in MGP expression is associated with reduced ROS levels in chondrocytes [6]. Our study demonstrated the role of UCMA in inhibiting ROS production in osteoblasts and in alleviating the impact of hyperglycemic stress. Osteoblasts treated with high glucose concentrations and UCMA displayed a drastic suppression of ROS production compared with that in osteoblasts treated with high glucose concentrations alone. Additionally, UCMA treatment increased the expression of Nrf2 and SOD1, causing significant changes in mitochondrial fission, but not in fusion; therefore, it is necessary to determine the exact molecular mechanism of UCMA. Furthermore, UCMA treatment inhibited the activation of the AKT signaling pathway. It has been previously shown that the FLAG tag on the UCMA protein did not change the function of UCMA or have any cellular effect [14]. In addition, the concentration of UCMA used in this study was first selected based on cell viability assays, which demonstrated that 5 μg/ml of UCMA reduced high glucose-induced ROS production. Nevertheless, the dose or duration of UCMA treatment in this study could have been sub-optimal, and further studies may be required.
HO-1 is an enzyme anchored in the endoplasmic reticulum and expressed in various intracellular compartments, including the mitochondria, nucleus, and plasma membrane [5]. Its translocation to the nucleus has been demonstrated in conditions associated with physiological stress, such as diabetes, neurodegenerative diseases, and inflammatory diseases [22, 35]. HO-1 is an oxidant-sensitive gene that is directly triggered by oxidative stress molecules, such as H2O2 and OH, and thiol-reactive substances, such as CdCl2 and NaAsO2 [21]. Moreover, HO-1 upregulation inhibits osteoblast mineralization and differentiation [16]. In this study, HO-1 was expressed in the nuclei of high glucose-treated osteoblasts and its nuclear expression was reduced in cells following UCMA addition. However, further investigations on the anti-oxidant effects of UCMA on osteoblasts treated directly with oxidative stress molecules or thiol-reactive substances may improve our knowledge of the other functions of UCMA.
Mitochondrial fission and fusion are essential for their normal function and determine their metabolic activity and ATP generation. Mitochondrial fission is proportional to the number of mitochondria in a cell and serves as a mechanism by which stress-damaged components are accumulated in daughter mitochondria and can be removed by autophagy [17, 27]. It also decreases membrane potential and causes autophagic removal of damaged cells [10]. Mitochondrial fusion minimizes mitochondrial dysfunction by decreasing the concentration of mutant mitochondrial genes and maintaining stable ATP production under stress conditions [40]. Excessive fission results in small mitochondrial fragments, whereas excessive fusion leads to the formation of elongated mitochondria. Mitochondrial fission and fusion are dynamically regulated by DRP1 and MFN2, respectively. DRP1-dependent fission is required in mitochondria during cell division and contributes to the elimination of damaged cells. Although alterations in mitochondrial fission were not directly observed in high glucose-treated osteoblasts with or without UCMA, DRP1 expression was increased in osteoblasts treated with high glucose levels and UCMA. However, the expression of proteins involved in mitochondrial dynamics under hyperglycemic conditions still remains controversial [12, 20], and further studies in this regard are warranted.
ROS activates the PI3K/AKT signaling pathway, which is essential for various cellular processes [39]. Hyperglycemia activates AKT signaling and induces MSC aging [38]. A previous study showed that OC reduces ROS production induced under hyperglycemic conditions through the AKT signaling pathway [18]. Our study showed the inhibitory activity of UCMA on the AKT signaling pathway activated by the high glucose-induced ROS production. Although we did not confirm the direct inhibitory role of UCMA by activation of AKT signaling under hyperglycemic conditions, a significant decrease in AKT signaling in cells treated with high glucose and UCMA suggests that UCMA is more effective in chronic hyperglycemia, although further research is required to determine this effect.
In conclusion, this study suggests a novel role for UCMA in elucidating the antioxidant effects of ameliorating hyperglycemic stress in osteoblasts.
Acknowledgment
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C1003398).
The Conflict of Interest Statement
The authors declare that they have no conflicts of interest with the contents of this article.
참고문헌
- Alikhani, M., Alikhani, Z., Boyd, C., MacLellan, C. M., Raptis, M., Liu, R., Pischon, N., Trackman, P. C., Gerstenfeld, L. and Graves, D. T. 2017. Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways. Bone 40, 345-353. https://doi.org/10.1016/j.bone.2006.09.011
- Amri, K., Freund, N., Vilar, J., Merlet-Benichou, C. and Lelievre-Pegorier, M. 1999. Adverse effects of hyperglycemia on kidney development in rats: in vivo and in vitro studies. Diabetes 48, 2240-2245. https://doi.org/10.2337/diabetes.48.11.2240
- Capulli, M., Paone, R. and Rucci, N. 2014. Osteoblast and osteocyte: games without frontiers. Arch. Biochem. Biophys. 561, 3-12. https://doi.org/10.1016/j.abb.2014.05.003
- Cunha, J. S., Ferreira, V. M., Maquigussa, E., Naves, M. A. and Boim, M. A. 2014. Effects of high glucose and high insulin concentrations on osteoblast function in vitro. Cell Tissue Res. 358, 249-256. https://doi.org/10.1007/s00441-014-1913-x
- Dunn, L. L., Midwinter, R. G., Ni, J., Hamid, H. A., Parish, C. R. and Stocker, R. 2014. New insights into intracellular locations and functions of heme oxygenase-1. Antioxid. Redox Signal. 20, 1723-1742 https://doi.org/10.1089/ars.2013.5675
- Ebrahiminezhad, A. and Sarabadani, Z. 2021. The impact of a key nutraceutical complex on chondrocyte cells and matrix Gla protein expression. Biocatal. Agric. Biotechnol. 32, 101930.
- Hao, Z., Jin, D. Y., Stafford, D. W. and Tie, J. K. 2019. Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study. Haematologica 105, 2164-2173. https://doi.org/10.3324/haematol.2019.229047
- Jafar, N., Edriss, H. and Nugent, K. 2016. The effect of short-term hyperglycemia on the innate immune system. Am. J. Med. Sci. 351, 201-211. https://doi.org/10.1016/j.amjms.2015.11.011
- Jiao, H., Xiao, E. and Graves, D. T. 2015. Diabetes and its effect on bone and fracture healing. Curr. Osteoporos. Rep. 13, 327-335. https://doi.org/10.1007/s11914-015-0286-8
- Kaikini, A. A., Kanchan, D. M., Nerurkar, U. N. and Sathaye, S. 2017. Targeting mitochondrial dysfunction for the treatment of diabetic complications: Pharmacological interventions through natural products. Pharmacogn. Rev. 11, 128-135. https://doi.org/10.4103/phrev.phrev_41_16
- Komori, T. 2006. Regulation of osteoblast differentiation by transcription factors. J. Cell. Biochem. 99, 1233-1239. https://doi.org/10.1002/jcb.20958
- Kumari, S., Anderson, L., Farmer, S., Mehta, S. L. and Li, P. A. 2012. Hyperglycemia alters mitochondrial fission and fusion proteins in mice subjected to cerebral ischemia and reperfusion. Transl. Stroke Res. 3, 296-304. https://doi.org/10.1007/s12975-012-0158-9
- Lee, S. H., Lee, Y. J., Park, S. I. and Kim, J. E. 2020. Unique cartilage matrix-associated protein inhibits the migratory and invasive potential of triple-negative breast cancer. Biochem. Biophys. Res. Commun. 530, 680-685. https://doi.org/10.1016/j.bbrc.2020.07.114
- Lee, Y. J., Park, S. Y., Lee, S. J., Boo, Y. C., Choi, J. Y. and Kim, J. E. 2015. Ucma, a direct transcriptional target of Runx2 and Osterix, promotes osteoblast differentiation and nodule formation. Osteoarthritis Cartilage 23, 1421-1431. https://doi.org/10.1016/j.joca.2015.03.035
- Lee, Y. J., Park, S. Y., Park, E. K. and Kim, J. E. 2019. Unique cartilage matrix-associated protein regulates fibrillin-2 expression and directly interacts with fibrillin-2 protein independent of calcium binding. Biochem. Biophys. Res. Commun. 511, 221-227. https://doi.org/10.1016/j.bbrc.2019.01.128
- Lin, T. H., Tang, C. H., Hung, S. Y., Liu, S. H., Lin, Y. M., Fu, W. M. and Yang, R. S. 2010. Upregulation of heme oxygenase-1 inhibits the maturation and mineralization of osteoblasts. J. Cell. Physiol. 222, 757-768. https://doi.org/10.1002/jcp.22008
- Lindner, A. B., Madden, R., Demarez, A., Stewart, E. J. and Taddei, F. 2008. Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc. Natl. Acad. Sci. USA 105, 3076-3081. https://doi.org/10.1073/pnas.0708931105
- Liu, J. and Yang, J. 2016. Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells. Int. J. Mol. Med. 37, 173-181. https://doi.org/10.3892/ijmm.2015.2412
- Mapanga, R. F. and Essop, M. F. 2016. Damaging effects of hyperglycemia on cardiovascular function: spotlight on glucose metabolic pathways. Am. J. Physiol. Heart Circ. Physiol. 310, H153-H173. https://doi.org/10.1152/ajpheart.00206.2015
- Pahwa, H., Khan, M. T. and Sharan, K. 2020. Hyperglycemia impairs osteoblast cell migration and chemotaxis due to a decrease in mitochondrial biogenesis. Mol. Cell. Biochem. 469, 109-118. https://doi.org/10.1007/s11010-020-03732-8
- Ryter, S. W. and Choi, A. M. 2005. Heme oxygenase-1: redox regulation of a stress protein in lung and cell culture models. Antioxid. Redox Signal. 7, 80-91. https://doi.org/10.1089/ars.2005.7.80
- Schipper, H. M. 1999. Glial HO-1 expression, iron deposition and oxidative stress in neurodegenerative diseases. Neurotox. Res. 1, 57-70. https://doi.org/10.1007/BF03033339
- Seuffert, F., Weidner, D., Baum, W., Schett, G. and Stock, M. 2018. Upper zone of growth plate and cartilage matrix associated protein protects cartilage during inflammatory arthritis. Arthritis Res. Ther. 20, 88.
- Stock, M. and Schett, G. 2021. Vitamin K-dependent proteins in skeletal development and disease. Int. J. Mol. Sci. 22, 9328.
- Surmann-Schmitt, C., Dietz, U., Kireva, T., Adam, N., Park, J., Tagariello, A., Onnerfjord, P., Heinegard, D., Schlotzer-Schrehardt, U., Deutzmann, R., von der Mark, K. and Stock, M. 2008. Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis. J. Biol. Chem. 283, 7082-7093. https://doi.org/10.1074/jbc.M702792200
- Tagariello, A., Luther, J., Streiter, M., Didt-Koziel, L., Wuelling, M., Surmann-Schmitt, C., Stock, M., Adam, N., Vortkamp, A. and Winterpacht, A. 2008. Ucma--A novel secreted factor represents a highly specific marker for distal chondrocytes. Matrix Biol. 27, 3-11. https://doi.org/10.1016/j.matbio.2007.07.004
- Tanaka, A., Cleland, M. M., Xu, S., Narendra, D. P., Suen, D. F., Karbowski, M. and Youle, R. J. 2010. Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. J. Cell Biol. 191, 1367-1380. https://doi.org/10.1083/jcb.201007013
- Tanaka, Y., Nakayamada, S. and Okada, Y. 2005. Osteoblasts and osteoclasts in bone remodeling and inflammation. Curr. Drug Targets Inflamm. Allergy 4 325-328. https://doi.org/10.2174/1568010054022015
- Thomas, D. M., Maher, F., Rogers, S. D. and Best, J. D. 1996. Expression and regulation by insulin of GLUT 3 in UMR 106-01, a clonal rat osteosarcoma cell line. Biochem. Biophys. Res. Commun. 218, 789-793. https://doi.org/10.1006/bbrc.1996.0140
- Viegas, C. S., Cavaco, S., Neves, P. L., Ferreira, A., Joao, A., Williamson, M. K., Price, P. A., Cancela, M. L. and Simes, D. C. 2008. Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates. J. Biol. Chem. 283, 36655-36664. https://doi.org/10.1074/jbc.M802761200
- Viegas, C. S., Simes, D. C., Laize, V., Williamson, M. K., Price, P.A. and Cancela, M. L. 2009. Gla-rich protein is a novel vitamin K-dependent protein present in serum that accumulates at sites of pathological calcifications. Am. J. Pathol. 175, 2288-2298. https://doi.org/10.2353/ajpath.2009.090474
- Wei, J., Shimazu, J., Makinistoglu, M. P., Maurizi, A., Kajimura, D., Zong, H., Takarada, T., Lezaki, T., Pessin, J. E., Hinoi, E. and Karsenty, G. 2015. Glucose uptake and Runx2 synergize to orchestrate osteoblast differentiation and bone formation. Cell 161, 1576-1591. https://doi.org/10.1016/j.cell.2015.05.029
- Willems, B. A., Furmanik, M., Caron, M. M. J., Chatrou, M. L. L., Kusters, D. H. M., Welting, T. J. M., Stock, M., Rafael, M. S., Viegas, C. S. B., Simes, D. C., Vermeer, C., Reutelingsperger, C. P. M. and Schurgers, L. J. 2018. Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling. Sci. Rep. 8, 4961.
- Wongdee, K. and Charoenphandhu, N. 2011. Osteoporosis in diabetes mellitus: Possible cellular and molecular mechanisms. World J. Diabetes 2, 41-48. https://doi.org/10.4239/wjd.v2.i3.41
- Xu, X., Li, H., Hou, X., Li, D., He, S., Wan, C., Yin, P., Liu, M., Liu, F. and Xu, J. 2015. Punicalagin induces Nrf2/HO-1 expression via upregulation of PI3K/AKT pathway and inhibits LPS-induced oxidative stress in RAW264.7 macrophages. Mediators Inflamm. 2015, 380218.
- Yu, T., Robotham, J. L. and Yoon, Y. 2006. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc. Natl. Acad. Sci. USA 103, 2653-2658. https://doi.org/10.1073/pnas.0511154103
- Yu, T., Sheu, S. S., Robotham, J. L. and Yoon, Y. 2008. Mitochondrial fission mediates high glucose-induced cell death through elevated production of reactive oxygen species. Cardiovasc. Res. 79, 341-351. https://doi.org/10.1093/cvr/cvn104
- Zhang, D., Lu, H., Chen, Z., Wang, Y., Lin, J., Xu, S., Zhang, C., Wang, B., Yuan, Z., Feng, X., Jiang, X. and Pan, J. 2017. High glucose induces the aging of mesenchymal stem cells via Akt/mTOR signaling. Mol. Med. Rep. 16, 1685-1690. https://doi.org/10.3892/mmr.2017.6832
- Zhao, Y., Hu, X., Liu, Y., Dong, S., Wen, Z., He, W., Zhang, S., Huang, Q. and Shi, M. 2017. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol. Cancer 16, 79.
- Zheng, C. X., Sui, B. D., Qiu, X. Y., Hu, C. H. and Jin, Y. 2020. Mitochondrial regulation of stem cells in bone homeostasis. Trends Mol. Med. 26, 89-104.