Rosa Adriana - Academia.edu (original) (raw)

Papers by Rosa Adriana

ABSTRACTWe previously showed the importance of TGFβ signaling in development of the mouse axial s... more ABSTRACTWe previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβR1 inhibitor, SB431542, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in SB431542 injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro- caudal markers within sclerotome was disrupted...

Biotechnology Progress, 2019

Reduced transforming growth factor beta (TGF‐β) signaling is associated with osteoarthritis (OA).... more Reduced transforming growth factor beta (TGF‐β) signaling is associated with osteoarthritis (OA). TGF‐β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF‐β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue‐engineered cartilage model with reduced TGF‐β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF‐β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF‐β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF‐β type I receptor inhibitor, SB431542, was used to ...

The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, 2008

Primary cilia are nonmotile microtubule-based appendages extending from the surface of almost all... more Primary cilia are nonmotile microtubule-based appendages extending from the surface of almost all vertebrate cells. The process of intraflagellar transport (IFT) is responsible for building and maintaining the structure and function of primary cilia. Disruption of Kif3a, a component of the Kinesin-II motor complex, disables anterograde IFT and leads to failure in the formation and maintenance of cilia. Likewise, the absence of IFT88/Tg737/Polaris, a core component of the IFT particle, results in the loss of cilia. Although primary cilia were described on chondrocytes almost 40 years ago, only recently has the functional significance of IFT and cilia in skeletal development been uncovered through the use of mouse models containing mutations or deletions in genes required to make and maintain cilia. Together, the results indicate that primary cilia/IFT are involved in coordinating multiple signaling pathways within the skeleton.

Journal of Bone and Mineral Research

Scientific Reports, 2020

Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome result... more Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-β regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-β stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-β did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-β-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-β-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-β treatment. Next, the role of several noncanonical TGF-β pathways were ...

Scientific Reports, 2016

Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key tr... more Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. TGF-β can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-β and that TGF-β stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-β. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-β. Here we show that p38 is required for TGF-β-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-β regulates Sox9 may lead to identification of therapeutic targets for OA. Articular cartilage is a connective tissue that provides a protective layer for the joints 1. Injury of this tissue can lead to a common condition called Osteoarthritis (OA) 2-4. Articular cartilage has limited repair properties. Successful therapeutic approaches to prevent damage or promote repair of cartilage have not been elucidated 5,6. For these reasons, new avenues potentially leading to disease modifying drugs need to be pursued. Previous studies identified important signaling pathways and transcription factors that are affected in OA. One of these, Transforming Growth Factor Beta (TGF-β) plays an important role in cartilage development and homeostasis 7,8. TGF-β signals through serine/threonine kinase receptors known as TGF-β type II (Tgfbr2) and type I (Tgfbr1). When TGF-β ligand binds to Tgfbr2 it recruits Tgfbr1 to form a heteromeric complex. Tgfbr2, a serine/threonine kinase, then phosphorylates Tgfbr1, activating the receptor, which then activates downstream targets 9,10. TGF-β can signal through what are considered canonical and non-canonical pathways 11. In the canonical pathway, Smad2 or Smad3 are phosphorylated by Tgfbr1. Phospho-Smad2 or 3 (pSmad2/3) then associate with Smad4 and translocate to the nucleus, bind to DNA, and regulate gene expression 10,12. In non-canonical signaling pathways, TGF-β activates MAPK kinase pathways including ERK, JNK, and p38, as well as the Rho-like GTPase, and phosphatidylinositol-3-kinase (PI3K)/AKT pathways 13. Previously, we showed that mice harboring a dominant negative mutation of Tgfbr2 (DNIIR) exhibited OA-like phenotype 14. Similar OA-like phenotype was shown in mice deficient in Smad3 and in adult rats with diminished p38 activity 15,16. Over-expression of TGF-β , can help in the repair of articular cartilage, through an increase in Collagen type II (Col2a) and Aggrecan (Acan) matrix, and inhibition of hypertrophic differentiation 17-20. However, increased levels of TGF-β can also lead to osteophyte formation exacerbating the OA phenotype 21. For this reason, downstream targets of TGF-β that specifically regulate chondroprotective pathways must be identified to develop preventative and reparative therapies. Sex determining region Y (SRY) Box 9 (Sox9) is an important chondrogenic transcription factor. It regulates formation of embryonic cartilage and is required for post-natal maintenance of the articular cartilage 22,23. Sox9

Journal of Biomechanics, 2014

Primary cilia are slender, microtubule based structures found in the majority of cell types with ... more Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88 fl/fl transgenic mice results in upregulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88 fl/fl transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage.

Developmental biology, 2007

In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of... more In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called “rotation” that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and postnatal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.

Annals of the New York Academy of Sciences, 2015

Primary cilia are microtubule-based organelles that project from the cell surface to enable trans... more Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes.

BMC developmental biology, 2010

Background: Very little is known about how intervertebral disc (IVD) is formed or maintained. Mem... more Background: Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-β superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing mouse tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-β has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-β action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing mouse IVD and to begin to understand the mechanism of TGF-β action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing mouse vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice as well as in sclerotome cultures treated with TGF-β or BMP4. Results: Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We also generated lists of genes regulated by TGF-β or BMP4 in cultured sclerotome. As expected, treatment with BMP4 resulted in up-regulation of cartilage marker genes including Acan, Sox 5, Sox6, and Sox9. In contrast, treatment with TGF-β1 did not regulate expression of cartilage markers but instead resulted in up-regulation of many IVD markers including Fmod and Adamtsl2. Conclusions: We propose TGF-β has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-β that warrant further investigation as regulators of IVD development.

Arthritis Research & Therapy, 2012

Introduction: Previous studies have indicated that transforming growth factor β (TGF-β) signaling... more Introduction: Previous studies have indicated that transforming growth factor β (TGF-β) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-β's chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-β that could act to maintain biochemical and biomechanical properties of cartilage. Methods: Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-β type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-β, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-β or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-β-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-β-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein. Results: DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-β. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-β and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice. Conclusion: TGF-β maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.

ABSTRACTWe previously showed the importance of TGFβ signaling in development of the mouse axial s... more ABSTRACTWe previously showed the importance of TGFβ signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFβ signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFβR1 inhibitor, SB431542, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in SB431542 injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFβ signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro- caudal markers within sclerotome was disrupted...

Biotechnology Progress, 2019

Reduced transforming growth factor beta (TGF‐β) signaling is associated with osteoarthritis (OA).... more Reduced transforming growth factor beta (TGF‐β) signaling is associated with osteoarthritis (OA). TGF‐β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF‐β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue‐engineered cartilage model with reduced TGF‐β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF‐β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF‐β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF‐β type I receptor inhibitor, SB431542, was used to ...

The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, 2008

Primary cilia are nonmotile microtubule-based appendages extending from the surface of almost all... more Primary cilia are nonmotile microtubule-based appendages extending from the surface of almost all vertebrate cells. The process of intraflagellar transport (IFT) is responsible for building and maintaining the structure and function of primary cilia. Disruption of Kif3a, a component of the Kinesin-II motor complex, disables anterograde IFT and leads to failure in the formation and maintenance of cilia. Likewise, the absence of IFT88/Tg737/Polaris, a core component of the IFT particle, results in the loss of cilia. Although primary cilia were described on chondrocytes almost 40 years ago, only recently has the functional significance of IFT and cilia in skeletal development been uncovered through the use of mouse models containing mutations or deletions in genes required to make and maintain cilia. Together, the results indicate that primary cilia/IFT are involved in coordinating multiple signaling pathways within the skeleton.

Journal of Bone and Mineral Research

Scientific Reports, 2020

Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome result... more Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-β regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-β stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-β did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-β-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-β-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-β treatment. Next, the role of several noncanonical TGF-β pathways were ...

Scientific Reports, 2016

Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key tr... more Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. TGF-β can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-β and that TGF-β stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-β. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-β. Here we show that p38 is required for TGF-β-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-β regulates Sox9 may lead to identification of therapeutic targets for OA. Articular cartilage is a connective tissue that provides a protective layer for the joints 1. Injury of this tissue can lead to a common condition called Osteoarthritis (OA) 2-4. Articular cartilage has limited repair properties. Successful therapeutic approaches to prevent damage or promote repair of cartilage have not been elucidated 5,6. For these reasons, new avenues potentially leading to disease modifying drugs need to be pursued. Previous studies identified important signaling pathways and transcription factors that are affected in OA. One of these, Transforming Growth Factor Beta (TGF-β) plays an important role in cartilage development and homeostasis 7,8. TGF-β signals through serine/threonine kinase receptors known as TGF-β type II (Tgfbr2) and type I (Tgfbr1). When TGF-β ligand binds to Tgfbr2 it recruits Tgfbr1 to form a heteromeric complex. Tgfbr2, a serine/threonine kinase, then phosphorylates Tgfbr1, activating the receptor, which then activates downstream targets 9,10. TGF-β can signal through what are considered canonical and non-canonical pathways 11. In the canonical pathway, Smad2 or Smad3 are phosphorylated by Tgfbr1. Phospho-Smad2 or 3 (pSmad2/3) then associate with Smad4 and translocate to the nucleus, bind to DNA, and regulate gene expression 10,12. In non-canonical signaling pathways, TGF-β activates MAPK kinase pathways including ERK, JNK, and p38, as well as the Rho-like GTPase, and phosphatidylinositol-3-kinase (PI3K)/AKT pathways 13. Previously, we showed that mice harboring a dominant negative mutation of Tgfbr2 (DNIIR) exhibited OA-like phenotype 14. Similar OA-like phenotype was shown in mice deficient in Smad3 and in adult rats with diminished p38 activity 15,16. Over-expression of TGF-β , can help in the repair of articular cartilage, through an increase in Collagen type II (Col2a) and Aggrecan (Acan) matrix, and inhibition of hypertrophic differentiation 17-20. However, increased levels of TGF-β can also lead to osteophyte formation exacerbating the OA phenotype 21. For this reason, downstream targets of TGF-β that specifically regulate chondroprotective pathways must be identified to develop preventative and reparative therapies. Sex determining region Y (SRY) Box 9 (Sox9) is an important chondrogenic transcription factor. It regulates formation of embryonic cartilage and is required for post-natal maintenance of the articular cartilage 22,23. Sox9

Journal of Biomechanics, 2014

Primary cilia are slender, microtubule based structures found in the majority of cell types with ... more Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88 fl/fl transgenic mice results in upregulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88 fl/fl transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage.

Developmental biology, 2007

In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of... more In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called “rotation” that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and postnatal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.

Annals of the New York Academy of Sciences, 2015

Primary cilia are microtubule-based organelles that project from the cell surface to enable trans... more Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes.

BMC developmental biology, 2010

Background: Very little is known about how intervertebral disc (IVD) is formed or maintained. Mem... more Background: Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-β superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing mouse tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-β has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-β action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing mouse IVD and to begin to understand the mechanism of TGF-β action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing mouse vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice as well as in sclerotome cultures treated with TGF-β or BMP4. Results: Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We also generated lists of genes regulated by TGF-β or BMP4 in cultured sclerotome. As expected, treatment with BMP4 resulted in up-regulation of cartilage marker genes including Acan, Sox 5, Sox6, and Sox9. In contrast, treatment with TGF-β1 did not regulate expression of cartilage markers but instead resulted in up-regulation of many IVD markers including Fmod and Adamtsl2. Conclusions: We propose TGF-β has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-β that warrant further investigation as regulators of IVD development.

Arthritis Research & Therapy, 2012

Introduction: Previous studies have indicated that transforming growth factor β (TGF-β) signaling... more Introduction: Previous studies have indicated that transforming growth factor β (TGF-β) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-β's chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-β that could act to maintain biochemical and biomechanical properties of cartilage. Methods: Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-β type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-β, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-β or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-β-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-β-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein. Results: DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-β. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-β and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice. Conclusion: TGF-β maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.