IU Indianapolis ScholarWorks :: Browsing by Subject "osteoblasts"

Browsing by Subject "osteoblasts"

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Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass
(Elsevier, 2019) Maupin, Kevin A.; Himes, Evan R.; Plett, Artur P.; Chua, Hui Lin; Singh, Pratibha; Ghosh, Joydeep; Mohamad, Safa F.; Abeysekera, Irushi; Fisher, Alexa; Sampson, Carol; Hong, Jung-Min; Childress, Paul; Alvarez, Marta; Srour, Edward F.; Bruzzaniti, Angela; Pelus, Louis M.; Orschell, Christie M.; Kacena, Melissa A.; Orthopaedic Surgery, School of Medicine
Osteoblast number and activity decreases with aging, contributing to the age-associated decline of bone mass, but the mechanisms underlying changes in osteoblast activity are not well understood. Here, we show that the age-associated bone loss critically depends on impairment of the ability of megakaryocytes (MKs) to support osteoblast proliferation. Co-culture of osteoblast precursors with young MKs is known to increase osteoblast proliferation and bone formation. However, co-culture of osteoblast precursors with aged MKs resulted in significantly fewer osteoblasts compared to co-culture with young MKs, and this was associated with the downregulation of transforming growth factor beta. In addition, the ability of MKs to increase bone mass was attenuated during aging as transplantation of GATA1low/low hematopoietic donor cells (which have elevated MKs/MK precursors) from young mice resulted in an increase in bone mass of recipient mice compared to transplantation of young wild-type donor cells, whereas transplantation of GATA1low/low donor cells from old mice failed to enhance bone mass in recipient mice compared to transplantation of old wild-type donor cells. These findings suggest that the preservation or restoration of the MK-mediated induction of osteoblast proliferation during aging may hold the potential to prevent age-associated bone loss and resulting fractures.
Differentiation and Activity of Murine Derived Stromal Osteoblasts After Electromagnetic Wave Stimulation
(2022) Wu, Jennifer L.; Spolnik, Kenneth; Bruzzaniti, Angela; Ehrlich, Ygal; Warner, Ned
Introduction: Elimination of bacteria and active infection within an infected root canal system is one of the primary objectives of nonsurgical root canal treatment. One of the measures of successful root canal treatment is subsequent bone healing of periapical lesions caused by previous infection. A previous study by Yumoto et al. showed that electromagnetic wave stimulation can increase proliferation of osteoblastic cells with no cytotoxicity, and it can also up-regulate growth factors such as vascular endothelial growth factor and platelet-derived growth factor.18 They also showed increased proliferation of an immortalized osteoblastic MC3T3-E1 cell line 3 days following electromagnetic stimulation (EMS).18 Previously, Pauly et al. found increased alkaline phosphatase (ALP) activity with 10 mA EMS application to primary murine calvaria-derived osteoblastic cells with 5 pulses at 1 second per pulse, but no significant differences were found for MTS proliferation nor mineral deposition compared to a negative control group.82 Optimization of the different variables including post-treatment incubation time, current delivery, and number of pulses per treatment may be necessary to improve osteogenic activity. The use of mesenchymal stem cells from murine bone marrow may also offer a physiologically relevant model for osteoblastic regeneration of periapical lesions. Objectives: The goal of this study was to investigate and optimize the effects of electromagnetic wave stimulation (EMS) on murine bone marrow mesenchymal stem cells (MSCs) by evaluating the proliferation and differentiation of the cells after exposure to different EMS treatment regimens. Materials and Methods: 5 x104 stromal osteoblasts (SOBs) were cultured in 24-well plates in α-MEM containing 10% fetal bovine serum. Cells were then subjected to pulsed EMS treatments of 1 mA, 10 mA, and 50 mA. EMS was generated using an electromagnetic apical treatment (EMAT) device created by J. Morita MFG Corp. Proliferation was assessed via MTS assay 1 days after treatment. For osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media, and SOBs were cultured for 14 days. Afterwards, alkaline phosphatase (ALP) activity and Alizarin-red S mineral deposition were quantified as measures of osteoblast activity. Cells grown in osteogenic media without EMS treatment served as the negative control. Results: Although MSC proliferation was unaffected by different EMS treatment regimens, 50 mA EMS resulted in a decrease in ALP activity and mineral deposition by osteoblasts. Conclusions: Our findings suggest bone healing by EMS may involve a different cellular mechanism, that is not reproduced in vitro in our studies. Utilizing different amperage and EMS regimens may improve osteogenic differentiation.
Kalirin Decreases Bone Mass Through Effects in Both Osteoclasts and Osteoblasts
(Office of the Vice Chancellor for Research, 2012-04-13) Huang, Su; Eleniste, Pierre; LeBlanc, Paula; Brown, Drew; Allen, Matthew R.; Bruzzaniti, Angela
Bone homeostasis is maintained by the balance between osteoclasts which degrade bone and osteoblasts, which form new bone. When the activity of either of these cells is dysregulated, bone loss can ensue, leading to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. The activity of osteoclasts and osteoblasts is regulated by local and systemic factors, as well as by key signaling proteins expressed in these cells. Kalirin is a novel GTP-exchange factor protein that plays a role in signaling pathways leading to cytoskeletal remodeling and dendritic spine formation in neurons, but its function in other cells is unknown. Western blotting and real time PCR confirmed that Kalirin is expressed in osteoclasts and osteoblasts, suggesting it may play a role in regulating bone cell function and bone mass. We used micro-CT to examine the bone phenotype of 14 week old female mice lacking Kalirin in all tissues (Kal-KO). Kal-KO mice exhibited a 40% lower trabecular bone volume in the distal femur compared to wild-type (WT) mice (n=9/group, p<0.05). We next quantified osteoclasts in histological sections by counting multinucleated cells expressing tartrate-resistant acid phosphatase (TRAP), a marker of mature osteoclasts. We found 48% higher osteoclast surface/bone surface in trabecular bone of Kal-KO mice, compared to WT mice (n=6/group, p<0.05). Osteoclast differentiation is controlled by osteoblasts, which secrete receptor activator of NF-kB ligand (RANKL), macrophage colony stimulating factor (MCSF) and osteoprotegerin (OPG), a decoy receptor for RANKL. We examined if Kalirin could regulate osteoclast differentiation in vitro. Osteoclasts were generated from the bone marrow of WT or Kal-KO mice by incubation with RANKL and MCSF for 7 days, and TRAP+ multinucleated cells were counted. Consistent with our in vivo studies, osteoclast number was significantly higher in cultures from Kal-KO mice, compared to WT mice. We next examined if Kalirin altered the ratio of secreted RANKL and OPG secreted by osteoblasts. Osteoblasts were generated from the calvaria of 2 day old neonates and the level of secreted RANKL and OPG in conditioned media was quantified by ELISA. Consistent with increased osteoclast differentiation, we found a higher RANKL/OPG ratio in conditioned media from Kal-KO osteoblasts, compared to WT cells. These data confirm a role for Kalirin in the regulation of trabecular bone mass through effects in both osteoclasts and osteoblasts.
Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts
(2012-05) Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela
The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.
Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress
(Office of the Vice Chancellor for Research, 2014-04-11) Sato, A; Plotkin, L; Bellido, T
Increased oxidative stress, such as with excess of glucocorticoids (GC) or during aging, has been associated with endoplasmic reticulum (ER) stress, due to accumulation of misfolded or unfolded proteins, leading to cellular apoptosis. The double-stranded RNA-activated protein kinase-like ER kinase (PERK) is activated to alleviate ER stress and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (eIF2α). Phosphorylated eIF2α in turn inhibits global protein translation to provide time to the ER to recover from the unfolded protein load, promoting cell viability. We hypothesized that the pro-apoptotic effect of GC on osteoblasts and osteocytes are at least in part due to induction of ER stress. To test this hypothesis, we used MLO-Y4 osteocytic cells, OB-6 osteoblastic cells, and primary osteoblastic cells derived from neonatal murine calvaria. We found that the synthetic GC dexamethasone (DEX) significantly increased the percentage of apoptotic cells in cultures of MLO-Y4, OB-6, and primary osteoblastic cells. Similarly, the specific ER-stress inducing agents brefeldin A, an inhibitor of ER-golgi apparatus vesicle transport, and tunicamycin, a protein glycosylation inhibitor, significantly increased OB-6 cell apoptosis. We then tested the effect of salubrinal, an agent that protects against ER stress by inhibiting the dephosphorylation of eIF2α, on bone cell apoptosis. Salubrinal blocked apoptosis induced by the ER stressors brefeldin A and tunicamycin in OB-6 cells. Salubrinal was also effective in blocking apoptosis induced by DEX in MLO-Y4, OB-6 and primary osteoblastic cells. Optimal responses were found at 10 μM salubrinal, after either 6 or 24 h. Guanabenz, another inhibitor of eIF2α dephosphorylation, also blocked DEX and tunicamycin-induced apoptosis of primary osteoblastic cells. Furthermore, addition of DEX to mineralizing OB-6 or primary osteoblastic cells markedly decreased mineral deposition and hydroxyapatite formation. In contrast, treatment with guanabenz increased mineralization of OB-6 cell cultures and prevented the inhibitory effect of DEX. We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells are mediated through ER stress and that interventions that prevent dephosphorylation of eIF2α could potentially prevent the deleterious effects of GC on bone.
Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice
(Elsevier, 2017-10) Withers, Catherine N.; Brown, Drew M.; Byiringiro, Innocent; Allen, Matthew R.; Condon, Keith W.; Satin, Jonathan; Andres, Douglas A.; Department of Anatomy and Cell Biology, School of Medicine
The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2 + channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad−/− calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, + 11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.
Selective inactivation of Stat3 in osteoclasts affect bone mass differently in female and male mice
(Office of the Vice Chancellor for Research, 2013-04-05) Himes, Evan; Zhou, Hongkang; Li, Jiliang
Signal Transducer and Activator of Transcription 3 (Stat3) is activated by the binding of various cytokines to their receptors, such as IL-6. Previous studies have revealed that conditional knockouts of Stat3 in osteoblasts and osteocytes cause a decrease in bone mineral density and strength. To study the role of Stat3 in osteoclasts, osteoclast- specific knockout mice were created using cre-lox recombination. Bone mineral density (BMD) and bone mineral content (BMC) were calculated for femurs and the fourth lumbar vertebra (L4) of 8 weeks old mice. Analysis revealed a decrease in BMD of femurs of osteoclast-selective Stat3 knockout (KOOc-Stat3) mice compared to their littermate control (p<0.05). There was also a decrease in BMC of the femurs of KOOc-Stat3 mice compared to the littermate controls (p<0.05). Analysis of μCT data from trabecular bone in the distal femur showed significant decreases in trabecular number and bone volume/tissue volume in both male and female KOOc-Stat3 mice. Trabecular separation was increased in male and female KOOc-Stat3 mice. Bone histomorphometry at the distal femur revealed a significant decrease in bone formation rate in males and females KOOc-Stat3 mice compared to the littermate controls. Osteoclast number identified by tartrate resistant acid phosphatase (TRAP) stain in female KOOc-Stat3 mice was significantly deficient from their control. These data suggest that inactivation of Stat3 in osteoclasts influences bone metabolism through both osteoblasts and osteoclasts. Knockout of Stat3 in either cell type leads to decreases in bone strength, making Stat3 a good drug target for treatment of diseases such as osteoporosis.
Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues
(Elsevier, 2021-01) Dasgupta, Krishnakali; Lessard, Samantha; Hann, Steven; Fowler, Megan A.; Robling, Alexander G.; Warman, Matthew L.; Anatomy and Cell Biology, School of Medicine
In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3caH1047R) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3caH1047R/+ offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3caH1047R/+ offspring survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3caH1047R/+ and Tg(DMP1-Cre);Pik3caH1047R/+ mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude that differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation are due to Cre-recombination being induced at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination.

Browsing by Subject "osteoblasts"

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