The Experts below are selected from a list of 1623 Experts worldwide ranked by ideXlab platform
Barbara D. Boyan - One of the best experts on this subject based on the ideXlab platform.
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The role of PDIA3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation
Journal of Immunology, 2016Co-Authors: Bianca Baker, Zvi Schwartz, Barbara D. Boyan, Brian O. Barnstein, Nicholas M. Clark, John J. RyanAbstract:Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (PDIA3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. PDIA3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking PDIA3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E 2 , which is released during PDIA3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE 2 , was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.
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A review of 1α,25(OH)2D3 dependent PDIA3 receptor complex components in Wnt5a non-canonical pathway signaling.
The Journal of steroid biochemistry and molecular biology, 2015Co-Authors: Maryam Doroudi, Rene Olivares-navarrete, Barbara D. Boyan, Zvi SchwartzAbstract:Wnt5a and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] regulate endochondral ossification. 1α,25(OH)2D3 initiates its calcium-dependent effects via its membrane-associated receptor, protein disulfide isomerase A3 (PDIA3). 1α,25(OH)2D3 binding to PDIA3 triggers the interaction between PDIA3 and phospholipase A2 (PLA2)-activating protein (PLAA), resulting in downstream activation of calcium/calmodulin-dependent protein kinase II (CaMKII), PLA2, and protein kinase C (PKC). Wnt5a initiates its calcium-dependent effects via binding its receptors Frizzled2 (FZD2) and Frizzled5 (FZD5) and receptor tyrosine kinase-like orphan receptor 2 (ROR2), activating intracellular calcium release and stimulating PKC and CaMKII. Recent efforts to determine the inter-relation between Wnt5a and 1α,25(OH)2D3 signaling pathways have demonstrated that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade in chondrocytes and osteoblasts in which the components of the PDIA3 receptor complex were required. Furthermore, ROR2, but not FZD2 or FZD5, was required to mediate the calcium-dependent actions of 1α,25(OH)2D3. This review provides evidence that 1α,25(OH)2D3 and Wnt5a mediate their calcium-dependent pathways via similar receptor components and proposes that these pathways may interact since they are competing for the same receptor complex components.
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Impaired Bone Formation in PDIA3 Deficient Mice
PloS one, 2014Co-Authors: Yun Wang, Christophe S.d. Lee, Alexandr Nizkorodov, Kelsie Riemenschneider, Zvi Schwartz, Rene Olivares-navarrete, Barbara D. BoyanAbstract:1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated PDIA3-deficient mice and examined the physiologic consequence of PDIA3-disruption in embryos and PDIA3+/− heterozygotes at different ages. No mice homozygous for the PDIA3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the PDIA3 gene resulted in early embryonic lethality. PDIA3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, PDIA3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in PDIA3+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in PDIA3+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from PDIA3+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the PDIA3+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.
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Membrane-mediated actions of 1,25-dihydroxy vitamin D3: A review of the roles of phospholipase A2 activating protein and Ca2+/calmodulin-dependent protein kinase II
The Journal of steroid biochemistry and molecular biology, 2014Co-Authors: Maryam Doroudi, Zvi Schwartz, Barbara D. BoyanAbstract:The secosteroid 1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. In its membrane-initiated pathway, after 1α,25(OH)2D3 interacts with protein disulfide isomerase, family A, member 3 (PDIA3) in caveolae, phospholipase A2 (PLA2) and protein kinase C (PKC) are activated. Recent efforts to determine the signaling proteins involved in the 1α,25(OH)2D3 signal from PDIA3 to PLA2 have indicated that phospholipase A2 activating protein (PLAA) and Ca2+/calmodulin-dependent kinase II (CaMKII) are required. PLAA is located in caveolae, where it interacts with PDIA3 and caveolin-1 (Cav-1) to initiate rapid signaling via CaMKII, activating PLA2, leading to activation of protein kinase C (PKC) and PKC-dependent responses.
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Membrane actions of 1α,25(OH)2D3 are mediated by Ca2+/calmodulin-dependent protein kinase II in bone and cartilage cells
The Journal of steroid biochemistry and molecular biology, 2014Co-Authors: Maryam Doroudi, Barbara D. Boyan, Marc C. Plaisance, Zvi SchwartzAbstract:1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (PDIA3) in caveolae. 1α,25(OH)2D3 binding to PDIA3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, PDIA3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or PDIA3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via PDIA3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3.
Zvi Schwartz - One of the best experts on this subject based on the ideXlab platform.
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The role of PDIA3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation
Journal of Immunology, 2016Co-Authors: Bianca Baker, Zvi Schwartz, Barbara D. Boyan, Brian O. Barnstein, Nicholas M. Clark, John J. RyanAbstract:Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (PDIA3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. PDIA3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking PDIA3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E 2 , which is released during PDIA3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE 2 , was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.
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A review of 1α,25(OH)2D3 dependent PDIA3 receptor complex components in Wnt5a non-canonical pathway signaling.
The Journal of steroid biochemistry and molecular biology, 2015Co-Authors: Maryam Doroudi, Rene Olivares-navarrete, Barbara D. Boyan, Zvi SchwartzAbstract:Wnt5a and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] regulate endochondral ossification. 1α,25(OH)2D3 initiates its calcium-dependent effects via its membrane-associated receptor, protein disulfide isomerase A3 (PDIA3). 1α,25(OH)2D3 binding to PDIA3 triggers the interaction between PDIA3 and phospholipase A2 (PLA2)-activating protein (PLAA), resulting in downstream activation of calcium/calmodulin-dependent protein kinase II (CaMKII), PLA2, and protein kinase C (PKC). Wnt5a initiates its calcium-dependent effects via binding its receptors Frizzled2 (FZD2) and Frizzled5 (FZD5) and receptor tyrosine kinase-like orphan receptor 2 (ROR2), activating intracellular calcium release and stimulating PKC and CaMKII. Recent efforts to determine the inter-relation between Wnt5a and 1α,25(OH)2D3 signaling pathways have demonstrated that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade in chondrocytes and osteoblasts in which the components of the PDIA3 receptor complex were required. Furthermore, ROR2, but not FZD2 or FZD5, was required to mediate the calcium-dependent actions of 1α,25(OH)2D3. This review provides evidence that 1α,25(OH)2D3 and Wnt5a mediate their calcium-dependent pathways via similar receptor components and proposes that these pathways may interact since they are competing for the same receptor complex components.
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Impaired Bone Formation in PDIA3 Deficient Mice
PloS one, 2014Co-Authors: Yun Wang, Christophe S.d. Lee, Alexandr Nizkorodov, Kelsie Riemenschneider, Zvi Schwartz, Rene Olivares-navarrete, Barbara D. BoyanAbstract:1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated PDIA3-deficient mice and examined the physiologic consequence of PDIA3-disruption in embryos and PDIA3+/− heterozygotes at different ages. No mice homozygous for the PDIA3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the PDIA3 gene resulted in early embryonic lethality. PDIA3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, PDIA3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in PDIA3+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in PDIA3+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from PDIA3+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the PDIA3+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.
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Membrane-mediated actions of 1,25-dihydroxy vitamin D3: A review of the roles of phospholipase A2 activating protein and Ca2+/calmodulin-dependent protein kinase II
The Journal of steroid biochemistry and molecular biology, 2014Co-Authors: Maryam Doroudi, Zvi Schwartz, Barbara D. BoyanAbstract:The secosteroid 1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. In its membrane-initiated pathway, after 1α,25(OH)2D3 interacts with protein disulfide isomerase, family A, member 3 (PDIA3) in caveolae, phospholipase A2 (PLA2) and protein kinase C (PKC) are activated. Recent efforts to determine the signaling proteins involved in the 1α,25(OH)2D3 signal from PDIA3 to PLA2 have indicated that phospholipase A2 activating protein (PLAA) and Ca2+/calmodulin-dependent kinase II (CaMKII) are required. PLAA is located in caveolae, where it interacts with PDIA3 and caveolin-1 (Cav-1) to initiate rapid signaling via CaMKII, activating PLA2, leading to activation of protein kinase C (PKC) and PKC-dependent responses.
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Membrane actions of 1α,25(OH)2D3 are mediated by Ca2+/calmodulin-dependent protein kinase II in bone and cartilage cells
The Journal of steroid biochemistry and molecular biology, 2014Co-Authors: Maryam Doroudi, Barbara D. Boyan, Marc C. Plaisance, Zvi SchwartzAbstract:1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (PDIA3) in caveolae. 1α,25(OH)2D3 binding to PDIA3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, PDIA3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or PDIA3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via PDIA3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3.
Duncan R. Smith - One of the best experts on this subject based on the ideXlab platform.
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Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression Proteins and proteomics
Biochimica et Biophysica Acta, 2019Co-Authors: Chontida Tangsongcharoen, Sittiruk Roytrakul, Duncan R. SmithAbstract:The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.
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Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression
Biochimica et biophysica acta. Proteins and proteomics, 2018Co-Authors: Chontida Tangsongcharoen, Sittiruk Roytrakul, Duncan R. SmithAbstract:The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.
Jiaxuan Chen - One of the best experts on this subject based on the ideXlab platform.
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New insights on membrane mediated effects of 1α,25-dihydroxy vitamin D3 signaling in the musculoskeletal system.
Steroids, 2013Co-Authors: Maryam Doroudi, Jiaxuan Chen, Barbara D. Boyan, Zvi SchwartzAbstract:Abstract 1α,25-Dihydroxy vitamin D3 [1α,25(OH) 2 D 3 ] acts on cells via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. Two receptors have been implicated to play roles in 1α,25(OH) 2 D 3 mediated rapid signaling, the classical nuclear vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (PDIA3). Long term efforts to investigate the roles of these two receptors demonstrated thatPDIA3 is located in caveolae, where it interacts with phospholipase A2 (PLA2) activating protein (PLAA) and caveolin-1 (Cav-1) to initiate rapid signaling via Ca ++ /calmodulin-dependent protein kinase II (CaMKII), PLA2, phospholipase C (PLC), protein kinase C (PKC), and ultimately the ERK1/2 family of mitogen activated protein kinases (MAPK). VDR is present on the plasma membrane, and it is required for 1α,25(OH) 2 D 3 induced rapid activation of Src. PDIA3+/− mice demonstrate an impaired musculoskeletal phenotype. Moreover, our studies examining mineralization of pre-osteoblasts in 3D culture have shown the physiological importance of PDIA3 and VDR interaction: knockdown of PDIA3 or VDR is characterized by impaired mineralization of the constructs.
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Plasma membrane PDIA3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3
Cellular signalling, 2013Co-Authors: Jiaxuan Chen, Zvi Schwartz, Maryam Doroudi, Jeffery Cheung, Ashley L. Grozier, Barbara D. BoyanAbstract:1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (PDIA3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)2D3. We found that PDIA3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both PDIA3 and VDR interacted with caveolin-1. PDIA3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)2D3 changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)2D3 regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)2D3 and subsequently regulating osteoblast biology.
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Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors
Journal of tissue engineering and regenerative medicine, 2013Co-Authors: Jiaxuan Chen, Barbara D. Boyan, Christopher R. Dosier, Jung Hwa Park, Robert E. Guldberg, Zvi SchwartzAbstract:1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (PDIA3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), PDIA3-silenced (Sh-PDIA3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(e-caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing PDIA3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either PDIA3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-PDIA3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and PDIA3. Copyright © 2013 John Wiley & Sons, Ltd.
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Chaperone properties of PDIA3 participate in rapid membrane actions of 1α,25-dihydroxyvitamin D3
Molecular endocrinology (Baltimore Md.), 2013Co-Authors: Jiaxuan Chen, Sharon L. Hyzy, Barbara D. Boyan, Rene Olivares-navarrete, Kirill S. Lobachev, Brian J. Grindel, Mary C. Farach-carson, Khairat Elbaradie, Maryam Doroudi, Zvi SchwartzAbstract:Protein disulfide isomerase family A, member 3 (PDIA3) mediates many of the plasma membrane (PM)-associated rapid responses to 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3). It is not well understood how PDIA3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1α,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for PDIA3's ER chaperone function, and examined their role in responses to 1α,25(OH)2D3. PDIA3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for PDIA3. Finally, we determined whether palmitoylation and/or myristoylation were required for PDIA3-mediated responses to 1α,25(OH)2D3. Overexpressing the PDIA3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1α,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overex...
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Mechanism of PDIA3-dependent 1α,25-dihydroxy vitamin D3 signaling in musculoskeletal cells.
Steroids, 2012Co-Authors: Barbara D. Boyan, Jiaxuan Chen, Zvi SchwartzAbstract:Abstract 1α,25-Dihydroxy vitamin D3 [1,25(OH)2D3] acts on cells through traditional steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-associated signaling pathways. Two receptors have been implicated in rapid signaling by 1,25(OH)2D3, the classical nuclear vitamin D receptor (VDR) and the more recently identified protein disulfide isomerase, family A, member 3 (PDIA3). Our lab along with other groups has established various tools to investigate the role of these two receptors, including gene knock-out, conditional knock-out, silencing, and over-expression in various model systems (growth plate chondrocytes, osteoblastic cells, chick intestinal epithelial cells, mouse embryoid bodies, extracellular matrix vesicles and isolated cell membranes). The data demonstrate the requirement for PDIA3 in 1,25(OH)2D3 induced phospholipase A2 (PLA2) and protein kinase C (PKC) activation and downstream responses. PDIA3+/− heterozygote mice also exhibit both cartilage and bone defects. VDR is present on the plasma membrane and one VDR−/− mouse strain lacks transcaltachia, although 1,25(OH)2D3 induced PKC activation and transcaltachia are not affected in another VDR−/− mouse strain. In the context of osteoblast differentiation, both receptors are expressed during osteogenic commitment of embryoid bodies and silencing of each causes a more mature osteoblast phenotype in MC3T3-E1 pre-osteoblasts. PDIA3 exists in caveolae, where it interacts with PLA2 activating protein (PLAA) and caveolin-1 to initiate rapid signaling via PLA2, phospholipase C (PLC), PKC, and ultimately the ERK1/2 family of mitogen activated protein kinases (MAPK). Using the growth plate chondrocyte and matrix vesicle models, we have demonstrated that PDIA3-dependent signaling in response to 1,25(OH)2D3 regulates growth plate physiology.
Chontida Tangsongcharoen - One of the best experts on this subject based on the ideXlab platform.
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Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression Proteins and proteomics
Biochimica et Biophysica Acta, 2019Co-Authors: Chontida Tangsongcharoen, Sittiruk Roytrakul, Duncan R. SmithAbstract:The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.
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Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression
Biochimica et biophysica acta. Proteins and proteomics, 2018Co-Authors: Chontida Tangsongcharoen, Sittiruk Roytrakul, Duncan R. SmithAbstract:The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.