Tamara Alliston

Publications of Tamara Alliston

• The use of polyacrylamide gels for mechanical calibration of cartilage--a combined nanoindentation and unconfined compression study.

  Authors: Cheng Li, Jessica Allen, Tamara Alliston, Lisa A Pruitt

  Journal of the mechanical behavior of biomedical materials. 10/2011; 4(7):1540-7.

  This study investigates polyacrylamide (PA) gel as a calibration material to measure the nanomechanical compressive modulus of cartilage using nanoindentation. Both nanoindentation and unconfinedThis study investigates polyacrylamide (PA) gel as a calibration material to measure the nanomechanical compressive modulus of cartilage using nanoindentation. Both nanoindentation and unconfined compression testing were performed on PA gel and porcine rib cartilage. The equilibrium moduli measured by the two methods were discernable. Nanoindentation has the advantage of distinguishing between spatially dependent constituent properties that affect tissue mechanical function in heterogeneous and hierarchically structured tissues such as cartilage. Both sets of measurements exhibited similar positive correlation with increasing gel crosslinker concentration. The compressive modulus measurements from compression in the PA gels ranged from 300 kPa-1.4 MPa, whereas those from nanoindentation ranged from 100 kPa-1.1 MPa. Using this data, a method for relating nanoindentation measurements to conventional mechanical property measurements is presented for porcine rib cartilage. It is shown that based on this relationship, the local tissue modulus as measured from nanoindentation (1.1-1.4 MPa) was able to predict the overall global modulus of the same sample of rib cartilage (2.2 MPa), as confirmed by experimental measurements from unconfined compression. This study supports the use of nanoindentation for the local characterization of cartilage tissues and may be applied to other soft tissues and constructs.

• Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone.

  Authors: Holly D Barth, Elizabeth A Zimmermann, Eric Schaible, Simon Y Tang, Tamara Alliston, Robert O Ritchie

  Biomaterials. 08/2011; 32(34):8892-904.

  Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties,Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ∼40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.

• Age-related changes in the plasticity and toughness of human cortical bone at multiple length scales.

  Authors: Elizabeth A Zimmermann, Eric Schaible, Hrishikesh Bale, Holly D Barth, Simon Y Tang, Peter Reichert, Bjoern Busse, Tamara Alliston, Joel W Ager, Robert O Ritchie

  Proceedings of the National Academy of Sciences of the United States of America. 08/2011; 108(35):14416-21.

  The structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeterThe structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, bone's toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural scales typically below a micrometer and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural scales. Biological factors such as aging lead to a markedly increased fracture risk, which is often associated with an age-related loss in bone mass (bone quantity). However, we find that age-related structural changes can significantly degrade the fracture resistance (bone quality) over multiple length scales. Using in situ small-angle X-ray scattering and wide-angle X-ray diffraction to characterize submicrometer structural changes and synchrotron X-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micrometer scales, we show how these age-related structural changes at differing size scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased nonenzymatic collagen cross-linking, which suppresses plasticity at nanoscale dimensions, and to an increased osteonal density, which limits the potency of crack-bridging mechanisms at micrometer scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by "plastic" deformation at higher structural levels, which occurs by the process of microcracking.

• Local tissue properties of human osteoarthritic cartilage correlate with magnetic resonance T(1) rho relaxation times.

  Authors: Simon Y Tang, Richard B Souza, Michael Ries, Paul K Hansma, Tamara Alliston, Xiaojuan Li

  Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 03/2011; 29(9):1312-9.

  The objective of this study is to examine the local relationship between T(1ρ) relaxation times and the mechanical behavior of human osteoarthritic articular cartilage using high-resolution magneticThe objective of this study is to examine the local relationship between T(1ρ) relaxation times and the mechanical behavior of human osteoarthritic articular cartilage using high-resolution magnetic resonance imaging (MRI) and local in situ microindentation. Seven human tibial plateaus were obtained from patients who underwent total knee arthroplasty due to severe osteoarthritis (OA). Three to six sites were selected from each sample for visual classification using the ICRS Outerbridge scale (a total of 36 sites). Samples were imaged by MR, and the local distribution of T(1ρ) relaxation times were obtained at these selected sites. The elastic and viscoelastic characteristics of the tissue were quantified nondestructively using dynamic microindentation to measure peak dynamic modulus, energy dissipation, and phase angle. Measured Outerbridge scores, MR T(1ρ) relaxation times, and mechanical properties were highly heterogeneous across each cartilage surface. Site-specific measures of T(1ρ) relaxation times correlated significantly with the phase angle (p < 0.001; R = 0.908), a viscoelastic mechanical behavior of the cartilage. The novel combination of high-resolution MR imaging and microindentation allows the investigation of the local relationship between quantitative MRI and biomechanical properties in highly heterogeneous OA cartilage. These findings suggest that MRI T(1ρ) can provide a functional assessment of articular cartilage.

• Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-β and Runx2 in bone is required for hearing.

  Authors: Jolie L Chang, Delia S Brauer, Jacob Johnson, Carol G Chen, Omar Akil, Guive Balooch, Mary Beth Humphrey, Emily N Chin, Alexandra E Porter, Kristin Butcher, Robert O Ritchie, Richard A Schneider, Anil Lalwani, Rik Derynck, Grayson W Marshall, Sally J Marshall, Lawrence Lustig, Tamara Alliston

  EMBO reports. 10/2010; 11(10):765-71.

  Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, includingPhysical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-β (TGFβ)-responsive pathway that controls osteoblast differentiation. Deregulated TGFβ or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFβ signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

• In situ Materials Characterization using the Tissue Diagnostic Instrument.

  Authors: Simon Y Tang, Phillip Mathews, Connor Randall, Eugene Yurtsev, Kirk Fields, Andrew Wong, Alfred Kuo, Tamara Alliston, Paul Hansma

  Polymer testing. 04/2010; 29(2):159-163.

  An understanding of the mechanical behavior of polymers is critical towards the design, implementation, and quality control of such materials. Yet experiments and method for the characterization ofAn understanding of the mechanical behavior of polymers is critical towards the design, implementation, and quality control of such materials. Yet experiments and method for the characterization of material properties of polymers remain challenging due the need to reconcile constitutive assumptions with experimental conditions. Well-established modes of mechanical testing, such as unconfined compression or uniaxial tension, require samples with specific geometries and carefully controlled orientations. Moreover, producing specimens that conform to such specifications often requires a considerable amount of sample material. In this study we validate a micromechanical indentation device, the Tissue Diagnostic Instrument (TDI), which implements a cyclic indentation method to determine the material properties of polymers and elastomeric materials. Measurements using the TDI require little or no sample preparation, and they allow the testing of sample materials in situ. In order to validate the use of the TDI, we compared measurements of modulus determined by the TDI to those obtained by unconfined compression tests and by uniaxial tension tests within the limit of small stresses and strains. The results show that the TDI measurements were significantly correlated with both unconfined compression (p<0.001; r(2) = 0.92) and uniaxial tension tests (p<0.001; r(2)=0.87). Moreover, the measurements across all three modes of testing were statistically indistinguishable from each other (p=0.92; ANOVA) and demonstrate that TDI measurements can provide a surrogate for the conventional methods of mechanical characterization.

• Osteopontin deficiency increases bone fragility but preserves bone mass.

  Authors: Philipp J Thurner, Carol G Chen, Sophi Ionova-Martin, Luling Sun, Adam Harman, Alexandra Porter, Joel W Ager, Robert O Ritchie, Tamara Alliston

  Bone. 02/2010; 46(6):1564-73.

  The ability of bone to resist catastrophic failure is critically dependent upon the material properties of bone matrix, a composite of hydroxyapatite, collagen type I, and noncollagenous proteins.The ability of bone to resist catastrophic failure is critically dependent upon the material properties of bone matrix, a composite of hydroxyapatite, collagen type I, and noncollagenous proteins. These properties include elastic modulus, hardness, and fracture toughness. Like other aspects of bone quality, matrix material properties are biologically-defined and can be disrupted in skeletal disease. While mineral and collagen have been investigated in greater detail, the contribution of noncollagenous proteins such as osteopontin to bone matrix material properties remains unclear. Several roles have been ascribed to osteopontin in bone, many of which have the potential to impact material properties. To elucidate the role of osteopontin in bone quality, we evaluated the structure, composition, and material properties of bone from osteopontin-deficient mice and wild-type littermates at several length scales. Most importantly, the results show that osteopontin deficiency causes a 30% decrease in fracture toughness, suggesting an important role for OPN in preventing crack propagation. This significant decline in fracture toughness is independent of changes in whole bone mass, structure, or matrix porosity. Using nanoindentation and quantitative backscattered electron imaging to evaluate osteopontin-deficient bone matrix at the micrometer level, we observed a significant reduction in elastic modulus and increased variability in calcium concentration. Matrix heterogeneity was also apparent at the ultrastructural level. In conclusion, we find that osteopontin is essential for the fracture toughness of bone, and reduced toughness in osteopontin-deficient bone may be related to the increased matrix heterogeneity observed at the micro-scale. By exploring the effects of osteopontin deficiency on bone matrix material properties, composition and organization, this study suggests that reduced fracture toughness is one mechanism by which loss of noncollagenous proteins contribute to bone fragility.

• Chondroitin sulfate and growth factor signaling in the skeleton: Possible links to MPS VI.

  Authors: Tamara Alliston

  Journal of pediatric rehabilitation medicine. 01/2010; 3(2):129-38.

  Mucopolysaccharidosis type VI (MPS VI), also called Maroteaux-Lamy syndrome, is an autosomal recessive lysosomal storage disorder caused by deficiency of a specific enzyme required forMucopolysaccharidosis type VI (MPS VI), also called Maroteaux-Lamy syndrome, is an autosomal recessive lysosomal storage disorder caused by deficiency of a specific enzyme required for glycosaminoglycan catabolism. Deficiency in the N-acetylgalactosamine-4-sulfatase (4S) enzyme, also called arylsulfatase B (ARSB), may have profound skeletal consequences. In MPS VI, partially degraded glycosaminoglycans (GAGs) such as dermatan sulfate and chondroitin sulfate accumulate within lysosomes. Through mechanisms that remain unclear, the abnormal GAG metabolism impacts several aspects of cellular function, particularly in the growth plate. This article explores the hypothesis that accrued partially degraded GAGs may contribute to deregulation of signaling pathways that normally orchestrate skeletal development, with a focus on members of the transforming growth factor-β (TGF-β) family. Understanding the molecular mechanisms disrupted by MPS VI may yield insight to improve the efficacy of MPS VI therapies, including bone marrow transplantation and enzyme replacement therapies.

• The tissue diagnostic instrument.

  Authors: Paul Hansma, Hongmei Yu, David Schultz, Azucena Rodriguez, Eugene A Yurtsev, Jessica Orr, Simon Tang, Jon Miller, Joseph Wallace, Frank Zok [......] Carol Chen, Mathilde Peters, David Kohn, Jenni Buckley, Xiaojuan Li, Lisa Pruitt, Adolfo Diez-Perez, Tamara Alliston, Valerie Weaver, Jeffrey Lotz

  The Review of scientific instruments. 06/2009; 80(5):054303.

  Tissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral diskTissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral disk degeneration, cancer, atherosclerosis, osteoarthritis, osteoporosis, and tooth decay. Here we introduce the tissue diagnostic instrument (TDI), a device designed to probe the mechanical properties of normal and diseased soft and hard tissues not only in the laboratory but also in patients. The TDI can distinguish between the nucleus and the annulus of spinal disks, between young and degenerated cartilage, and between normal and cancerous mammary glands. It can quantify the elastic modulus and hardness of the wet dentin left in a cavity after excavation. It can perform an indentation test of bone tissue, quantifying the indentation depth increase and other mechanical parameters. With local anesthesia and disposable, sterile, probe assemblies, there has been neither pain nor complications in tests on patients. We anticipate that this unique device will facilitate research on many tissue systems in living organisms, including plants, leading to new insights into disease mechanisms and methods for their early detection.

• The tissue diagnostic instrument

  Authors: Paul Hansma, Hongmei Yu, David Schultz, Eugene A. Yurtsev, Jessica Orr, Simon Tang, Jon Miller, Frank Zok, Richard Souza, Alexander Proctor, Xavier Nogues-Solan, M.Jesus Peña, Lisa Pruitt, Adolfo Diez-Perez, Tamara Alliston, Valerie Weaver, Jeff Lotz

  Review of Scientific Instruments. 05/2009;

  Tissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebralTissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral disk degeneration, cancer, atherosclerosis, osteoarthritis, osteoporosis, and tooth decay. Here we introduce the tissue diagnostic instrument �TDI�, a device designed to probe the mechanical properties of normal and diseased soft and hard tissues not only in the laboratory but also in patients. The TDI can distinguish between the nucleus and the annulus of spinal disks, between young and degenerated cartilage, and between normal and cancerous mammary glands. It can quantify the elastic modulus and hardness of the wet dentin left in a cavity after excavation. It can perform an indentation test of bone tissue, quantifying the indentation depth increase and other mechanical parameters. With local anesthesia and disposable, sterile, probe assemblies, there has been neither pain nor complications in tests on patients. We anticipate that this unique device will facilitate research on many tissue systems in living organisms, including plants, leading to new insights into disease mechanisms and methods for their early detection. © 2009 American Institute of Physics. �DOI: 10.1063/1.3127602� I. INTRODUCTION The tissue diagnostic instrument �TDI� was redesigned from the bone diagnostic instrument1,2 so as to measure tissue mechanical properties subcutaneously and in vivo with additional probe assemblies and an adjustable compliance �Fig. 1�. It consists of a thin probe assembly that can penetrate skin and soft tissue to reach deep tissues. The disposable, sterilizable probe assembly consists of an outer reference probe made from a 23 gauge hypodermic needle and an inner test probe made from stainless steel wire ranging from 175 to 300 �m in diameter and from 2 to 90 mm in length. Since friction between the test probe and the reference probe increases with length, it is desirable to use only the length needed to access the desired tissue location. The test probe is held in a nickel tube that couples to a magnet, which in turn is linked to a force generator. During operation the force generator oscillates the probe within the tissue of interest and concurrently measures the force and displacement. The maximum values for force and displacement are 12 N and 600 �m. The probe is typically operated at a frequency of 4 Hz because this is rapid enough to allow hand holding yet sufficiently slow to allow easy decoupling of the elastic and viscous response of the tissue �see supplementary material3 for more details including force and displacement ranges.� II. MEASUREMENTS We first illustrate TDI use in human spinal disks that are composed of a thick outer ligament �annulus fibrosus� and a central swelling hydrogel �nucleus pulposus�. Spinal disk degeneration can be the underlying cause of back pain leading to significant morbidity and societal expense. Intervertebral disks are one of the most highly loaded tissues in the body, and consequently material property insufficiency can lead to damage accumulation, inflammation, and pain. Disk degen- REVIEW OF SCIENTIFIC INSTRUMENTS 80, 054303 �2009� 0034-6748/2009/80�5�/054303/6/$25.00 80, 054303-1 © 2009 American Institute of Physics Downloaded 28 May 2009 to 72.215.163.35. Redistribution subject to AIP license or copyright; see http://rsi.aip.org/rsi/copyright.jsp

• A tense situation: forcing tumour progression.

  Authors: Darci T Butcher, Tamara Alliston, Valerie M Weaver

  Nature reviews. Cancer. 03/2009; 9(2):108-22.

  Cells within tissues are continuously exposed to physical forces including hydrostatic pressure, shear stress, and compression and tension forces. Cells dynamically adapt to force by modifying theirCells within tissues are continuously exposed to physical forces including hydrostatic pressure, shear stress, and compression and tension forces. Cells dynamically adapt to force by modifying their behaviour and remodelling their microenvironment. They also sense these forces through mechanoreceptors and respond by exerting reciprocal actomyosin- and cytoskeletal-dependent cell-generated force by a process termed 'mechanoreciprocity'. Loss of mechanoreciprocity has been shown to promote the progression of disease, including cancer. Moreover, the mechanical properties of a tissue contribute to disease progression, compromise treatment and might also alter cancer risk. Thus, the changing force that cells experience needs to be considered when trying to understand the complex nature of tumorigenesis.

• Pharmacologic inhibition of the tgf-Beta type I receptor kinase has anabolic and anti-catabolic effects on bone.

  Authors: Khalid S Mohammad, Carol G Chen, Guive Balooch, Elizabeth Stebbins, C Ryan McKenna, Holly Davis, Maria Niewolna, Xiang Hong Peng, Daniel H. N. Nguyen, Sophi S Ionova-Martin, John W. Bracey, William R Hogue, Darren H Wong, Robert O Ritchie, Larry J Suva, Rik Derynck, Theresa A Guise, Tamara Alliston

  PLoS ONE. 02/2009; 4(4):e5275.

  During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentallyDuring development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-beta has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-beta signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-beta signaling on bone remain unclear. To examine the role of TGF-beta in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-beta type I receptor (TbetaRI) kinase on bone mass, architecture and material properties. Inhibition of TbetaRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TbetaRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TbetaRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TbetaRI inhibitors may be effective in treating conditions of skeletal fragility.

• Elevated TGF-beta2 signaling in dentin results in sex related enamel defects.

  Authors: Kuniko Saeki, Joan F Hilton, Tamara Alliston, Stefan Habelitz, Sally J Marshall, Grayson W Marshall, Pamela Denbesten

  Archives of oral biology. 09/2007; 52(9):814-21.

  Initiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. In this study, we used a transgenic mouse model which drives overexpression of anInitiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. In this study, we used a transgenic mouse model which drives overexpression of an activated form of TGF-beta2 under control of the osteocalcin promoter, to investigate the role of TGF-beta2 in the dental mesenchyme, on enamel formation. Dentin and enamel were imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Dentin mechanical properties were characterized for hardness and elasticity, following nanoindentation with a modified AFM. Pores found in enamel were quantified and compared using image analysis software (Scion Imagetrade mark). The elastic modulus of dentin was significantly reduced in the male TGF-beta2 overexpressor mice as compared to male wildtype mice, with no significant differences between female mice. Similarly, there were significantly more pores in enamel of the male transgenic mice as compared to male wildtype mice, with no significant differences between female mice. In situ hybridization of the continuously erupting incisor confirmed that osteocalcin expression was limited to the odontoblast cell layer at all stages of tooth formation. TGF-beta2 overexpression in the dentin matrix, results in sex-linked differences in dentin and enamel formation.

• TGF-beta regulates the mechanical properties and composition of bone matrix.

  Authors: Guive Balooch, Mehdi Balooch, Ravi K Nalla, Stephen Schilling, Ellen H Filvaroff, Grayson W Marshall, Sally J Marshall, Robert O Ritchie, Rik Derynck, Tamara Alliston

  Proceedings of the National Academy of Sciences of the United States of America. 01/2006; 102(52):18813-8.

  The characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition ofThe characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition of bone matrix, along with bone mass and architecture, are critical determinants of a bone's ability to resist fracture. Several regulators of bone mass and architecture have been identified, but factors that regulate the mechanical properties and composition of bone matrix are largely unknown. We used a combination of high-resolution approaches, including atomic-force microscopy, x-ray tomography, and Raman microspectroscopy, to assess the properties of bone matrix independently of bone mass and architecture. Properties were evaluated in genetically modified mice with differing levels of TGF-beta signaling. Bone matrix properties correlated with the level of TGF-beta signaling. Smad3+/- mice had increased bone mass and matrix properties, suggesting that the osteopenic Smad3-/- phenotype may be, in part, secondary to systemic effects of Smad3 deletion. Thus, a reduction in TGF-beta signaling, through its effector Smad3, enhanced the mechanical properties and mineral concentration of the bone matrix, as well as the bone mass, enabling the bone to better resist fracture. Our results provide evidence that bone matrix properties are controlled by growth factor signaling.

• Repression of Runx2 function by TGF-beta through recruitment of class II histone deacetylases by Smad3.

  Authors: Jong Seok Kang, Tamara Alliston, Rachel Delston, Rik Derynck

  The EMBO journal. 08/2005; 24(14):2543-55.

  Transforming growth factor-beta (TGF-beta) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-beta/Smad3 inhibits Runx2Transforming growth factor-beta (TGF-beta) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-beta/Smad3 inhibits Runx2 function has not been characterized. We show that TGF-beta induces histone deacetylation, primarily of histone H4, at the osteocalcin promoter, which is repressed by TGF-beta, and that histone deacetylation is required for repression of Runx2 by TGF-beta. This repression occurs through the action of the class IIa histone deacetylases (HDAC)4 and 5, which are recruited through interaction with Smad3 to the Smad3/Runx2 complex at the Runx2-binding DNA sequence. Accordingly, HDAC4 or 5 is required for efficient TGF-beta-mediated inhibition of Runx2 function and is involved in osteoblast differentiation. Our results indicate that class IIa HDACs act as corepressors for TGF-beta/Smad3-mediated transcriptional repression of Runx2 function in differentiating osteoblasts and are cell-intrinsic regulators of osteoblast differentiation.

• Repression of bone morphogenetic protein and activin-inducible transcription by Evi-1.

  Authors: Tamara Alliston, Tien C Ko, Yanna Cao, Yao-Yun Liang, Xin-Hua Feng, Chenbei Chang, Rik Derynck

  The Journal of biological chemistry. 07/2005; 280(25):24227-37.

  Smads, key effectors of transforming growth factor (TGF)-beta, activin, and bone morphogenetic protein (BMP) signaling, regulate gene expression and interact with coactivators and corepressors thatSmads, key effectors of transforming growth factor (TGF)-beta, activin, and bone morphogenetic protein (BMP) signaling, regulate gene expression and interact with coactivators and corepressors that modulate Smad activity. The corepressor Evi-1 exerts its oncogenic effects by repressing TGF-beta/Smad3-mediated transcription, thereby blocking TGF-beta-induced growth arrest. Because Evi-1 interacts with the highly conserved MH2 domain of Smad3, we investigated the physical and functional interaction of Evi-1 with Smad1 and Smad2, downstream targets of BMP and activin signaling, respectively. Evi-1 interacted with and repressed the receptor-activated transcription through Smad1 and Smad2, similarly to Smad3. In addition, Evi-1 repressed BMP/Smad1- and activin/Smad2-mediated induction of endogenous Xenopus gene expression, suggesting a role of repression of BMP and activin signals by Evi-1 in vertebrate embryogenesis. Evi-1 also repressed the induction of endogenous Smad7 expression by TGF-beta family ligands. In the course of these studies, we observed Evi-1 repression of Smad transactivation even when Smad binding to DNA was kept constant. We therefore explored the mechanism of Evi-1 repression of TGF-beta family-inducible transcription. Evi-1 repression did not result from displacement of Smad binding to DNA or to CREB-binding protein but from the recruitment of Evi-1 by Smad3 and CREB-binding protein to DNA. Following TGF-beta stimulation, Evi-1 and the associated corepressor CtBP were recruited to the endogenous Smad7 promoter. Evi-1 recruitment to the promoter decreased TGF-beta-induced histone acetylation, coincident with its repression of Smad7 gene expression. In this way, Evi-1 acts as a general Smad corepressor to inhibit TGF-beta-, activin-, and BMP-inducible transcription.

• Transforming growth factor-beta 1 regulation of collagenase-3 expression in osteoblastic cells by cross-talk between the Smad and MAPK signaling pathways and their components, Smad2 and Runx2.

  Authors: Nagarajan Selvamurugan, Sukyee Kwok, Tamara Alliston, Michael Reiss, Nicola C Partridge

  The Journal of biological chemistry. 05/2004; 279(18):19327-34.

  Transforming growth factor-beta (TGF-beta) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblastsTransforming growth factor-beta (TGF-beta) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblasts and seems to be involved in osteoclastic bone resorption. Here, we show that TGF-beta 1 stimulates collagenase-3 expression in the rat osteoblastic cell line UMR 106-01 and requires de novo protein synthesis. Dominant-negative Smad2/3 constructs indicated that Smad signaling is essential for TGF-beta 1-stimulated collagenase-3 promoter activity. Inhibitors of the ERK1/2 and p38 MAPK pathways, but not the JNK pathway, reduced TGF-beta 1-stimulated collagenase-3 expression, indicating that the p38 MAPK and ERK1/2 pathways are also required for TGF-beta 1-stimulated collagenase-3 expression in UMR 106-01 cells. These inhibitors did not prevent nuclear localization of Smad proteins, but they inhibited Smad-mediated transcriptional activation. We have shown for the first time that Runx2 (a bone transcription factor and a potential substrate for the MAPK pathway) is phosphorylated in response to TGF-beta 1 treatment in osteoblastic cells. Cotransfection of Smad2 and Runx2 constructs had a cooperative effect on TGF-beta 1-stimulated collagenase-3 promoter activity in these cells. We further identified ligand-independent physical interaction between Smad2 and Runx2. Taken together, our results provide an important role for cross-talk between the Smad and MAPK pathways and their components in expression of collagenase-3 following TGF-beta 1 treatment in UMR 106-01 cells.

• Medicine: interfering with bone remodelling.

  Authors: Tamara Alliston, Rik Derynck

  Nature. 05/2002; 416(6882):686-7.