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Temple University
1.
Hendesi, Honey.
CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS.
Degree: PhD, 2014, Temple University
URL: http://digital.library.temple.edu/u?/p245801coll10,263674
► Cell Biology
Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates…
(more)
▼ Cell Biology
Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF…
Advisors/Committee Members: Popoff, Steven N., Barbe, Mary F., Rizzo, Victor, Goldfinger, Lawrence, Safadi, Fayez F., Owen, Thomas A.;.
Subjects/Keywords: Cellular biology;
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APA (6th Edition):
Hendesi, H. (2014). CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,263674
Chicago Manual of Style (16th Edition):
Hendesi, Honey. “CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS.” 2014. Doctoral Dissertation, Temple University. Accessed January 17, 2021.
http://digital.library.temple.edu/u?/p245801coll10,263674.
MLA Handbook (7th Edition):
Hendesi, Honey. “CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS.” 2014. Web. 17 Jan 2021.
Vancouver:
Hendesi H. CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS. [Internet] [Doctoral dissertation]. Temple University; 2014. [cited 2021 Jan 17].
Available from: http://digital.library.temple.edu/u?/p245801coll10,263674.
Council of Science Editors:
Hendesi H. CONNECTIVE TISSUE GROWTH FACTOR (CTGF/CCN2) REGULATES OSTEOBLAST CYTOSKELETAL REORGANIZATION AND MOTILITY AND ENHANCES DIFFERENTIATION VIA BINDING TO INTEGRIN RECEPTORS AND ACTIVATION OF DOWNSTREAM SIGNALINGS. [Doctoral Dissertation]. Temple University; 2014. Available from: http://digital.library.temple.edu/u?/p245801coll10,263674

Temple University
2.
Belcher, Joyce Yvonne.
Bone Cell Autonomous Effects of Osteoactivin In Vivo.
Degree: PhD, 2012, Temple University
URL: http://digital.library.temple.edu/u?/p245801coll10,183061
► Cell Biology
Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by…
(more)
▼ Cell Biology
Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix…
Advisors/Committee Members: Popoff, Steven N., Safadi, Fayez F., Barbe, Mary F., Monroy, Alexandra M., Yingling, Vanessa, Owen, Thomas A..
Subjects/Keywords: Cellular biology; Animal Model; Gpnmb; Osteoactivin; Osteoblasts; Osteoclasts; Osteopetrosis
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
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to Zotero / EndNote / Reference
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APA (6th Edition):
Belcher, J. Y. (2012). Bone Cell Autonomous Effects of Osteoactivin In Vivo. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,183061
Chicago Manual of Style (16th Edition):
Belcher, Joyce Yvonne. “Bone Cell Autonomous Effects of Osteoactivin In Vivo.” 2012. Doctoral Dissertation, Temple University. Accessed January 17, 2021.
http://digital.library.temple.edu/u?/p245801coll10,183061.
MLA Handbook (7th Edition):
Belcher, Joyce Yvonne. “Bone Cell Autonomous Effects of Osteoactivin In Vivo.” 2012. Web. 17 Jan 2021.
Vancouver:
Belcher JY. Bone Cell Autonomous Effects of Osteoactivin In Vivo. [Internet] [Doctoral dissertation]. Temple University; 2012. [cited 2021 Jan 17].
Available from: http://digital.library.temple.edu/u?/p245801coll10,183061.
Council of Science Editors:
Belcher JY. Bone Cell Autonomous Effects of Osteoactivin In Vivo. [Doctoral Dissertation]. Temple University; 2012. Available from: http://digital.library.temple.edu/u?/p245801coll10,183061

Temple University
3.
Singh, Maneet.
TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS.
Degree: PhD, 2011, Temple University
URL: http://digital.library.temple.edu/u?/p245801coll10,135232
► Cell Biology
Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression.…
(more)
▼ Cell Biology
Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation.
Temple University – Theses
Advisors/Committee Members: Safadi, Fayez F., Popoff, Steven N., Barbe, Mary F., Sanjay, Archana, Monroy, Alexandra M., Owen, Thomas A..
Subjects/Keywords: Cellular Biology; Molecular Biology; Biology; HOMEDOMAIN PROTEIN; OSTEOACTIVIN; OSTEOBLAST DIFFERENTIATION; RUNX2; SMAD1 AND SMAD4; TRANSCRIPTIONAL REGULATION
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Singh, M. (2011). TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,135232
Chicago Manual of Style (16th Edition):
Singh, Maneet. “TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS.” 2011. Doctoral Dissertation, Temple University. Accessed January 17, 2021.
http://digital.library.temple.edu/u?/p245801coll10,135232.
MLA Handbook (7th Edition):
Singh, Maneet. “TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS.” 2011. Web. 17 Jan 2021.
Vancouver:
Singh M. TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS. [Internet] [Doctoral dissertation]. Temple University; 2011. [cited 2021 Jan 17].
Available from: http://digital.library.temple.edu/u?/p245801coll10,135232.
Council of Science Editors:
Singh M. TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTS. [Doctoral Dissertation]. Temple University; 2011. Available from: http://digital.library.temple.edu/u?/p245801coll10,135232

Temple University
4.
Zhang, Xuemei.
Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts.
Degree: PhD, 2010, Temple University
URL: http://digital.library.temple.edu/u?/p245801coll10,63095
► Anatomy
Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and…
(more)
▼ Anatomy
Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular
matrix protein that acts as an anabolic growth factor to regulate osteoblast
differentiation and function. In osteoblasts, CTGF is induced by transforming growth
factor beta 1 (TGF-β1) where it acts as a downstream mediator of TGF-β1 induced
extracellular matrix production. The molecular mechanisms that control CTGF induction
by TGF-β1 in osteoblasts are not understood. We have previously demonstrated the
requirement of Src, Erk and Smad signaling for TGF-β1 induced CTGF promoter activity
in primary osteoblasts, however the potential interaction among these signaling
pathways in osteoblasts remains unknown. In this study, we demonstrate that CTGF is
induced by TGF-β1 in rat osteosarcoma osteoblast like cells (ROS17/2.8). TGF-β1
activates Src and blocking of Src family kinases by PP2 abrogates TGF-β1 induced
CTGF up-regulation. Western blot analysis revealed that primary osteoblasts and ROS
17/2.8 cells express not only Src, but also other Src family members, such as Fyn, Yes
and Hck. In order to determine whether CTGF up-regulation is controlled by Src or
other members, we used either kinase-dead dominant negative Src constructs in
primary osteoblasts or Src siRNA in ROS17/2.8 cells to block Src function. Inactivation
of Src by both kinase-dead and siRNA prevented TGF-β1 induced CTGF induction,
demonstrating that TGF-β1 induced CTGF up-regulation is mediated only by Src not by
other members. In addition, we also demonstrated that Erk is activated by TGF-β1 and
that blocking of Erk activation using pharmacological inhibitors, PD98059 and U0126,
prevents TGF-β1 induced CTGF induction, demonstrating the requirement of Erk for
CTGF induction. These results prompted us to further explore the cross-talk between
Src, Erk and Smads in ROS17/2.8 cells.
Inhibition of Src using PP2 prevented Erk activation, demonstrating that Src is
upstream of Erk. To investigate how Src and Erk regulate the canonical TGF-β1
signaling pathway, including Smad2/3 phosphorylation and nuclear translocation of
activated Smads, we treated cells with TGF-β1 in the presence or absence of the Src
inhibitor, PP2, or the Erk inhibitors, PD98059 or U0126. PP2 pre-treatment prevented
the phosphorylation of Smad2/3 at both the SSXS motif and the linker region and
consequently blocked their nuclear translocation, demonstrating that Src can regulate
Smad signaling. In contrast, the Erk inhibitors did not have any effects on Smad
phosphorylation and/or nuclear translocation. To examine whether Erk can modulate
Smad signaling indirectly through the activation/ inactivation of required nuclear coactivators/
co-repressors that mediate Smad DNA binding, we used electro-mobility shift
assays. These experiments showed that inhibition of Erk activation impaired
transcriptional complex formation on the Smad binding element (SBE) and TGF- β
responsive element (TRE) of the CTGF promoter, demonstrating that Erk activation is required
for SBE and TRE transactivation. Taking together,…
Advisors/Committee Members: Popoff, Steven N., Barbe, Mary F., Owen, Thomas A., Safadi, Fayez F., Sanjay, Archana.
Subjects/Keywords: Biology; Cell
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Zhang, X. (2010). Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,63095
Chicago Manual of Style (16th Edition):
Zhang, Xuemei. “Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts.” 2010. Doctoral Dissertation, Temple University. Accessed January 17, 2021.
http://digital.library.temple.edu/u?/p245801coll10,63095.
MLA Handbook (7th Edition):
Zhang, Xuemei. “Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts.” 2010. Web. 17 Jan 2021.
Vancouver:
Zhang X. Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts. [Internet] [Doctoral dissertation]. Temple University; 2010. [cited 2021 Jan 17].
Available from: http://digital.library.temple.edu/u?/p245801coll10,63095.
Council of Science Editors:
Zhang X. Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts. [Doctoral Dissertation]. Temple University; 2010. Available from: http://digital.library.temple.edu/u?/p245801coll10,63095
.