Publications by authors named "William A Horton"

36 Publications

Norms for Clinical Use of CXM, a Real-Time Marker of Height Velocity.

J Clin Endocrinol Metab 2021 Jan;106(1):e255-e264

Research Center, Shriners Hospitals for Children, Portland, Oregon.

Context: Height velocity (HV) is difficult to assess because growth is very slow. The current practice of calculating it from measurements taken at several-month intervals is insufficient for managing children with growth disorders. We identified a bone growth by-product (collagen X biomarker, CXM) in blood that in preliminary analysis in healthy children correlated strongly with conventionally determined HV and displayed a pattern resembling published norms for HV vs age.

Objective: The goal was to confirm our initial observations supporting the utility of CXM as an HV biomarker in a larger number of individuals and establish working reference ranges for future studies.

Design, Settings, And Participants: CXM was assessed in archived blood samples from 302 healthy children and 10 healthy adults yielding 961 CXM measurements. A total of 432 measurements were plotted by age, and sex-specific reference ranges were calculated. Serial values from 116 participants were plotted against observed HV. Matched plasma, serum, and dried blood spot readings were compared.

Results: A correlation of blood CXM with conventional HV was confirmed. Scatter plots of CXM vs age showed a similar pattern to current HV norms, and CXM levels demarcated the pubertal growth spurt both in girls and boys. CXM levels differed little in matched serum, plasma, and dried blood spot samples.

Conclusions: Blood CXM offers a potential means to estimate HV in real time. Our results establish sex-specific, working reference ranges for assessing skeletal growth, especially over time. CXM stability in stored samples makes it well suited for retrospective studies.
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http://dx.doi.org/10.1210/clinem/dgaa721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765635PMC
January 2021

A quantitative serum biomarker of circulating collagen X effectively correlates with endochondral fracture healing.

J Orthop Res 2021 01 20;39(1):53-62. Epub 2020 Jul 20.

Department of Orthopaedic Surgery, Orthopaedic Trauma Institute, Zuckerberg San Francisco General Hospital (ZSFG), University of California, San Francisco (UCSF), San Francisco, California.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm") and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.
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http://dx.doi.org/10.1002/jor.24776DOI Listing
January 2021

A degradation fragment of type X collagen is a real-time marker for bone growth velocity.

Sci Transl Med 2017 Dec;9(419)

Research Center, Shriners Hospitals for Children, Portland, OR 97239, USA.

Despite its importance as a key parameter of child health and development, growth velocity is difficult to determine in real time because skeletal growth is slow and clinical tools to accurately detect very small increments of growth do not exist. We report discovery of a marker for skeletal growth in infants and children. The intact trimeric noncollagenous 1 (NC1) domain of type X collagen, the marker we designated as CXM for Collagen X Marker, is a degradation by-product of endochondral ossification that is released into the circulation in proportion to overall growth plate activity. This marker corresponds to the rate of linear bone growth at time of measurement. Serum concentrations of CXM plotted against age show a pattern similar to well-established height growth velocity curves and correlate with height growth velocity calculated from incremental height measurements in this study. The CXM marker is stable once collected and can be accurately assayed in serum, plasma, and dried blood spots. CXM testing may be useful for monitoring growth in the pediatric population, especially responses of infants and children with genetic and acquired growth disorders to interventions that target the underlying growth disturbances. The utility of CXM may potentially extend to managing other conditions such as fracture healing, scoliosis, arthritis, or cancer.
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http://dx.doi.org/10.1126/scitranslmed.aan4669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516194PMC
December 2017

Advances in treatment of achondroplasia and osteoarthritis.

Hum Mol Genet 2016 Apr 6;25(R1):R2-8. Epub 2015 Oct 6.

Research Center, Shriners Hospital for Children, Portland, OR, USA and Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA

Achondroplasia (ACH) is the prototype and most common of the human chondrodysplasias. It results from gain-of-function mutations that exaggerate the signal output of the fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase that negatively regulates growth plate activity and linear bone growth. Several approaches to reduce FGFR3 signaling by blocking receptor activation or inhibiting downstream signals have been proposed. Five show promise in preclinical mouse studies. Two candidate therapies target the extracellular domain of FGFR3. The first is a decoy receptor that competes for activating ligands. The second is a synthetic blocking peptide that prevents ligands from binding and activating FGFR3. Two established drugs, statins and meclozine, improve growth of ACH mice. The strongest candidate therapy employs an analog of C-type natriuretic peptide (CNP), which antagonizes the mitogen-activated-protein (MAP) kinase pathway downstream of the FGFR3 receptor and may also act independently in the growth plate. Only the CNP analog has reached clinical trials. Preliminary results of Phase 2 studies show a substantial increase in growth rate of ACH children after six months of therapy with no serious adverse effects. A challenge for drug therapy in ACH is targeting agents to the avascular growth plate. The application of gene therapy in osteoarthritis offers insights because it faces similar technical obstacles. Major advances in gene therapy include the emergence of recombinant adeno-associated virus as the vector of choice, capsid engineering to target vectors to specific tissues, and development of methods to direct vectors to articular chondrocytes.
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http://dx.doi.org/10.1093/hmg/ddv419DOI Listing
April 2016

FGFR3 biology and skeletal disease.

Connect Tissue Res 2015 Nov 29;56(6):427-33. Epub 2015 Jul 29.

a Shriners Research Center, Shriners Hospitals for Children, Oregon Health & Science University , Portland , OR , USA.

Fibroblast Growth Factor Receptor 3 (FGFR3) is one of four high-affinity receptors for canonical FGF ligands. It acts in many tissues and plays a special role in skeletal development, especially post-embryonic bone growth, where it inhibits chondrocyte proliferation and differentiation. Gain of function mutations cause the most common forms of dwarfism in humans, and they are also detected in cancer. Triggered by ligand binding or in some cases mutation, FGFR3 activation involves dimerization of receptor monomers, phosphorylation of specific tyrosine residues in the receptor's kinase domain and in the tightly linked scaffold protein Fibroblast Receptor Factor Substrate 2 (FRS2). Signaling molecules recruited to these phosphorylation sites propagate signals through cascades that are subject to modulation. Signal output is also regulated by the fate of the receptor and the interval between its activation and degradation. Trafficking pathways have been identified for both lysosomal and proteasomal degradation, as well as, an alternative fate that involves intramembrane cleavage that produces an intracellular domain fragment capable of nuclear transport and potential function.
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http://dx.doi.org/10.3109/03008207.2015.1051224DOI Listing
November 2015

Correlation of the same fields imaged in the TEM, confocal, LM, and microCT by image registration: from specimen preparation to displaying a final composite image.

Methods Cell Biol 2014 ;124:391-417

Research Center, Shriners Hospital for Children, Portland, Oregon, USA.

Correlated imaging is the process of imaging a specimen with two complementary modalities and then registering and overlaying the fields obtained in each modality to create a composite view. One of the images is made somewhat transparent, allowing detail in the underlying image to be visible and assisting in the registration of the two images. As an example, an image localizing a specific tissue component by fluorescence may be overlaid atop a TEM image of the same field. The resulting composite image would demonstrate specific ultrastructural features in the high-resolution TEM field, which are colorized in the overlay. Other examples include composites from MicroCT or soft X-ray images overlaid atop light microscopy or TEM images. Automated image registration may be facilitated by a variety of sophisticated computer programs utilized by high-throughput laboratories. This chapter is meant for the more occasional user wishing to align images manually. ImageJ is a public domain, image processing program developed at the National Institutes of Health and is available to anyone as a free download. ImageJ performs marvelously well for the purpose of image registration; therefore, step-by-step instructions are included here. Specimen handling, including fixation and choice of embedding media, is not straightforward for correlative imaging. A step-by-step description of the protocols which work in our laboratory is included for simultaneous localization in LM, EM and micro-CT, as well as maintaining GFP emission in tissue embedded for TEM.
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http://dx.doi.org/10.1016/B978-0-12-801075-4.00018-5DOI Listing
June 2015

The paracrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and PTHrP in prehypertrophic chondrocytes.

J Cell Physiol 2014 Dec;229(12):1999-2014

Faculty of Veterinary Medicine, Department of Clinical Sciences of Companion Animals, Utrecht University, Utrecht, The Netherlands.

The endocrine feedback loop between vitamin D3(1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH-related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10(-8) M 1,25(OH)2D3 or PTHrP, Col2-pd2EGFP transgenic mice, and primary Col2-pd2EGFP growth plate chondrocytes isolated by FACS, using RT-qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α- and 24-hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration.
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http://dx.doi.org/10.1002/jcp.24658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298802PMC
December 2014

FGFR3 targeting strategies for achondroplasia.

Expert Rev Mol Med 2012 Jan 19;14:e11. Epub 2012 Jan 19.

Shriners Hospital for Children, Research Center, Portland, OR, USA.

Mutations that exaggerate signalling of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) give rise to achondroplasia, the most common form of dwarfism in humans. Here we review the clinical features, genetic aspects and molecular pathogenesis of achondroplasia and examine several therapeutic strategies designed to target the mutant receptor or its signalling pathways, including the use of kinase inhibitors, blocking antibodies, physiologic antagonists, RNAi and chaperone inhibitors. We conclude by discussing the challenges of treating growth plate disorders in children.
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http://dx.doi.org/10.1017/erm.2012.4DOI Listing
January 2012

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3.

Mol Biol Cell 2011 Oct 24;22(20):3861-73. Epub 2011 Aug 24.

Research Center, Shriners Hospital for Children, Portland, OR 97239, USA.

Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.
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http://dx.doi.org/10.1091/mbc.E11-01-0080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192865PMC
October 2011

The A391E mutation enhances FGFR3 activation in the absence of ligand.

Biochim Biophys Acta 2011 Aug 22;1808(8):2045-50. Epub 2011 Apr 22.

Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.

The A391E mutation in the transmembrane domain of fibroblast growth factor receptor 3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid stabilizes the dimeric receptor due to hydrogen bonding and enhances its ligand-independent activation. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated transmembrane domain dimer by -1.3°kcal/mol. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length fibroblast growth factor receptor 3 in human embryonic kidney 293T cells in the absence of ligand. We find that the mutation enhances the ligand-independent activation propensity of the receptor by -1.7°kcal/mol. This value is consistent with the observed strength of hydrogen bonds in membranes, and supports the above hypothesis.
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http://dx.doi.org/10.1016/j.bbamem.2011.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110564PMC
August 2011

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation.

J Biol Chem 2011 Jun 12;286(22):19597-604. Epub 2011 Apr 12.

Research Center, Shriners Hospital for Children, Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.
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http://dx.doi.org/10.1074/jbc.M110.206151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103339PMC
June 2011

Novel type II collagen reporter mice: New tool for assessing collagen 2α1 expression in vivo and in vitro.

Dev Dyn 2011 Mar 8;240(3):663-73. Epub 2011 Feb 8.

We report the generation of a new mouse strain harboring a Col2-pd2EGFP reporter transgene; pd2EGFP has a much shorter half-life than EGFP, making it a near real-time reporter for Col2α1 expression in vivo and in vitro. In the post-natal growth plate, pd2EGFP fluorescence was expressed in almost all proliferative chondrocytes and in some hypertrophic chondrocytes based on localization with type X collagen. In articular cartilage, pd2EGFP fluorescence diminished over time, nicely illustrating the decrease of type II collagen synthesis in articular chondrocytes during growth. Monolayers of FACS-sorted chondrocytes from P1-2 mice showed faster loss of pd2EGFP compared to EGFP, reflecting rapid chondrocyte de-differentiation. High-density culture of FACS-pd2EGFP- growth plate chondrocytes revealed the typical temporal expression pattern in which type II collagen preceded type X collagen matrix deposition. The Col2-pd2EGFP reporter mouse will be a valuable tool for studies of growth plate chondrocyte biology.
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http://dx.doi.org/10.1002/dvdy.22569DOI Listing
March 2011

Achondroplasia: pathogenesis and implications for future treatment.

Curr Opin Pediatr 2010 Aug;22(4):516-23

Research Center, Shriners Hospital for Children, Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.

Purpose Of Review: Although the genetic defect underlying achondroplasia has been known for over a decade, no effective therapies to stimulate bone growth have emerged. Here we review the recent literature and summarize the molecular mechanisms underlying disease pathology and examine their potential as therapeutic targets. Currently used preclinical models are discussed in the context of recent advances with a special focus on C-type natriuretic peptide.

Recent Findings: Research on the mutation in Fibroblast Growth Factor Receptor 3 (FGFR3) that causes achondroplasia suggests that disease results from increased signal transduction from the mutant receptor. Thus, current therapeutic strategies have focused on reducing signals emanating from FGFR3. First-generation therapies directly targeting FGFR3, such as kinase inhibitors and neutralizing antibodies, designed for targeting FGFR3 in cancer, are still in the preclinical phase and have yet to translate into the management of achondroplasia. Counteracting signal transduction pathways downstream of FGFR3 holds promise with the discovery that administration of C-type natriuretic peptide to achondroplastic mice ameliorates their clinical phenotype. However, more research into long-term effectiveness and safety of this strategy is needed. Direct targeting of therapeutic agents to growth plate cartilage may enhance efficacy and minimize side effects of these and future therapies.

Summary: Current research into the pathogenesis of achondroplasia has expanded our understanding of the mechanisms of FGFR3-induced disease and has increased the number of approaches that we may use to potentially correct it. Further research is needed to validate these approaches in preclinical models of achondroplasia.
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http://dx.doi.org/10.1097/MOP.0b013e32833b7a69DOI Listing
August 2010

FGFs in endochondral skeletal development.

J Cell Biochem 2010 Aug;110(5):1046-57

Shriners Hospital and Molecular & Medical Genetics and Cell & Developmental Biology, Oregon Health & Sciences University, Portland, Oregon 97239, USA.

The mammalian skeleton developments and grows through two complementary pathways: membranous ossification, which gives rise to the calvarial bones and distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face and base of the skull and the medial clavicle. Fibroblast growth factors (FGFs) and their cognate FGF receptors (FGFRs) play important roles in regulating both pathways. However, the details of how FGF signals are initiated, propagated and modulated within the developing skeleton are only slowly emerging. This prospect will focus on the current understanding of these events during endochondral skeletal development with special attention given to concepts that have emerged in the past few years.
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http://dx.doi.org/10.1002/jcb.22629DOI Listing
August 2010

Cartilage oligomeric matrix protein promotes cell attachment via two independent mechanisms involving CD47 and alphaVbeta3 integrin.

Mol Cell Biochem 2010 May 24;338(1-2):215-24. Epub 2009 Dec 24.

Medical Genetics Institute, Steven Spielberg Pediatric Research Center, Los Angeles, CA 90048, USA.

Cartilage oligomeric matrix protein (COMP) is a pentameric approximately 524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and alphaVbeta3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and alphaVbeta3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes.
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http://dx.doi.org/10.1007/s11010-009-0355-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150962PMC
May 2010

Crude subcellular fractionation of cultured mammalian cell lines.

BMC Res Notes 2009 Dec 10;2:243. Epub 2009 Dec 10.

Research Center, Shriners Hospitals for Children, Portland, Oregon 97239, USA.

Background: The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples.

Findings: We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step.

Conclusion: We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.
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http://dx.doi.org/10.1186/1756-0500-2-243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802353PMC
December 2009

FGFs in endochondral skeletal development.

Trends Endocrinol Metab 2009 Sep 27;20(7):341-8. Epub 2009 Aug 27.

Research Center, Shriners Hospital for Children, and Molecular & Medical Genetics, Oregon Health & Sciences University, Portland, OR 97239, USA.

The mammalian skeleton forms and grows through two developmental pathways: membranous ossification, which gives rise to calvarial bones and the distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face, base of the skull and the medial clavicle. The regulation of both pathways is extremely complex, and the rules that govern it are still emerging. However, it has become clear that fibroblast growth factors (FGFs) and their cognate receptors (FGFRs) play essential roles. This review focuses on the roles of FGFs and FGFRs in endochondral skeletal development, with special attention given to concepts that have emerged in the past few years.
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http://dx.doi.org/10.1016/j.tem.2009.04.003DOI Listing
September 2009

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Microsc Microanal 2008 Aug;14(4):342-8

Research Department, Shriners Hospital for Children, Portland, OR 97239, USA.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.
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http://dx.doi.org/10.1017/S1431927608080306DOI Listing
August 2008

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway.

Int J Biochem Cell Biol 2008 5;40(11):2649-59. Epub 2008 Jun 5.

Division of Biochemistry, Department of Morphological and Biomedical Sciences, University of Verona Medical School, Verona, Italy.

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.
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http://dx.doi.org/10.1016/j.biocel.2008.05.017DOI Listing
October 2008

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia.

Cell Signal 2008 Aug 10;20(8):1471-7. Epub 2008 Apr 10.

Research Center, Shriners Hospital for Children, 3101 S.W. Sam Jackson Park Road, Portland, OR 97239, USA.

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.
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http://dx.doi.org/10.1016/j.cellsig.2008.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675614PMC
August 2008

Achondroplasia.

Lancet 2007 Jul;370(9582):162-172

Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX, USA.

Achondroplasia is the most common form of short limb dwarfism in human beings, affecting more than 250,000 individuals worldwide. More than 95% of patients have the same point mutation in the gene for fibroblast growth factor receptor 3 (FGFR3) and more than 80% of these are new mutations. The mutation, which causes gain of FGFR3 function, affects many tissues, most strikingly the cartilaginous growth plate in the growing skeleton, leading to a variety of manifestations and complications. The biology of FGFR3 and the molecular and cellular consequences of the achondroplasia mutation are being elucidated, providing a more complete understanding of the disorder and a basis for future treatments targeted directly at relevant pathogenetic pathways. Furthermore, the natural history of the condition, which has been well delineated in childhood and adolescence, is being defined more fully in adults with achondroplasia; most of the serious complications can be modified favourably or prevented by anticipation and early treatment. Possible future treatments include chemical inhibition of receptor signalling, antibody blockade of receptor activation, and alteration of pathways that modulate the downstream propagation of FGFR3 signals.
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http://dx.doi.org/10.1016/S0140-6736(07)61090-3DOI Listing
July 2007

International workshop on the Skeletal Growth Plate Stevenson, Washington, June 11-15, 2006.

Matrix Biol 2007 May 20;26(4):324-9. Epub 2007 Feb 20.

Research Center, Shriners Hospital for Children, Oregon Health & Science University, Portland, Oregon, United States.

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http://dx.doi.org/10.1016/j.matbio.2007.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1992437PMC
May 2007

Developmental biology in orthopaedics. Summary of the 2006 AAOS research symposium.

J Bone Joint Surg Am 2007 Mar;89(3):668-71

Division of Orthopaedic Surgery and Program in Developmental Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada.

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http://dx.doi.org/10.2106/JBJS.F.01609DOI Listing
March 2007

BMP action in skeletogenesis involves attenuation of retinoid signaling.

J Cell Biol 2006 Jul;174(1):101-13

Department of Physiology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

The bone morphogenetic protein (BMP) and growth and differentiation factor (GDF) signaling pathways have well-established and essential roles within the developing skeleton in coordinating the formation of cartilaginous anlagen. However, the identification of bona fide targets that underlie the action of these signaling molecules in chondrogenesis has remained elusive. We have identified the gene for the retinoic acid (RA) synthesis enzyme Aldh1a2 as a principal target of BMP signaling; prochondrogenic BMPs or GDFs lead to attenuation of Aldh1a2 expression and, consequently, to reduced activation of the retinoid signaling pathway. Consistent with this, antagonism of retinoid signaling phenocopies BMP4 action, whereas RA inhibits the chondrogenic stimulatory activity of BMP4. BMP4 also down-regulates Aldh1a2 expression in organ culture and, consistent with this, Aldh1a2 is actively excluded from the developing cartilage anlagens. Collectively, these findings provide novel insights into BMP action and demonstrate that BMP signaling governs the fate of prechondrogenic mesenchyme, at least in part, through regulation of retinoid signaling.
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http://dx.doi.org/10.1083/jcb.200604150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064168PMC
July 2006

Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling.

J Bone Miner Res 2006 Apr 5;21(4):626-36. Epub 2006 Apr 5.

Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2579, USA.

Unlabelled: A novel role for IGF-I in MSC chondrogenesis was determined. IGF-I effects were evaluated in the presence or absence of TGF-beta signaling by conditionally inactivating the TGF-beta type II receptor. We found that IGF-I had potent chondroinductive actions on MSCs. IGF-I effects were independent from and additive to TGF-beta.

Introduction: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. TGF-beta induces MSC chondrogenic differentiation, although its actions are not well defined. The aim of our study was to define the biological role of IGF-I on proliferation, chondrogenic condensation, apoptosis, and differentiation of MSCs into chondrocytes, alone or in combination with TGF-beta and in the presence or absence of TGF-beta signaling.

Materials And Methods: Mononuclear adherent stem cells were isolated from mouse BM. Chondrogenic differentiation was induced by culturing high-density MSC pellets in serum- and insulin-free defined medium up to 7 days, with or without IGF-I and/or TGF-beta. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. Seven-day-old pellets were measured in size, stained for proteoglycan synthesis, and analyzed for the expression of collagen II and Sox-9 by quantitative real time PCR. We obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. We conditionally inactivated the TGF-beta type II receptor (TbetaRII) in MSCs using a cre-lox system, generating TbetaRII knockout MSCs (RIIKO-MSCs).

Results And Conclusions: IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. IGF-I chondroinductive actions were equally potent to TGF-beta1, and the two growth factors had additive effects. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-beta signaling. We found that the extracellular signal-related kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-beta1 mitogenic response and in part the IGF-I proliferative action. Our data, by showing the role of IGF-I and TGF-beta1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders.
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http://dx.doi.org/10.1359/jbmr.051213DOI Listing
April 2006

Recent milestones in achondroplasia research.

Authors:
William A Horton

Am J Med Genet A 2006 Jan;140(2):166-9

Research Center, Shriners Hospital for Children, Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, Oregon 97221, USA.

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http://dx.doi.org/10.1002/ajmg.a.31029DOI Listing
January 2006

Secretion of cartilage oligomeric matrix protein is affected by the signal peptide.

J Biol Chem 2005 Apr 3;280(17):17172-9. Epub 2005 Mar 3.

Research Center, Shriners Hospitals for Children, Portland, Oregon 97239, USA.

Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein found in the extracellular matrices of skeletal tissues. Mutations associated with two human skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, disturb COMP secretion leading to intracellular accumulation of mutant COMP, especially in chondrocytes. Here we show that the manifestation of this secretory defect is dramatically influenced by the signal peptide that targets COMP for secretion. The comparison of wild type and mutant COMP secretion directed by the COMP or BM40 signal peptide in HEK-293 cells and rat chondrosarcoma cells revealed that the BM40 signal peptide substantially enhances secretion of mutant COMP that accumulates in endoplasmic reticulum-like structures when targeted by its own signal peptide. Additionally, we demonstrate that mutant COMP forms mixed pentamers with wild type COMP. Our findings suggest that the secretory defect in pseudoachondroplasia and multiple epiphyseal dysplasia is not specific for chondrocytes, nor does it require interaction of mutant COMP with other matrix proteins prior to transport from the cell. They also imply a previously unappreciated role for the signal peptide in the regulation of protein secretion beyond targeting to the endoplasmic reticulum.
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http://dx.doi.org/10.1074/jbc.M411716200DOI Listing
April 2005

Targeted expression of SHH affects chondrocyte differentiation, growth plate organization, and Sox9 expression.

J Bone Miner Res 2004 Oct 12;19(10):1678-88. Epub 2004 Jul 12.

Department of Oncology, Biology and Genetics, University of Genoa Medical School, Genoa, Italy.

Unlabelled: The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.

Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates.

Materials And Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization.

Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter.

Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.
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http://dx.doi.org/10.1359/JBMR.040706DOI Listing
October 2004

Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence.

J Biomed Opt 2004 Mar-Apr;9(2):254-8

Oregon Health and Science University, Department of Biomedical Engineering and Dermatology, Shriners Hospital for Crippled Children, Portland, Oregon 97239, USA.

Optically monitoring the expression of green fluorescent protein (GFP) in the cartilage underlying the skin of a mouse allows tracking the expression of the chondrocyte phenotype. This paper considers how confocal microscopy with spectral detection can sense GFP fluorescence in the cartilage despite light scattering and collagen autofluorescence from the overlying skin. An in vivo experiment tested the abilities of a topical optical fiber measurement and a confocal microscope measurement to detect GFP in cartilage under the skin versus the collagen autofluorescence. An ex vivo experiment tested the ability of a confocal microscope without and with its pinhole to detect a fluorescent microsphere underneath an ex vivo skin layer versus the collagen autofluorescence. In both systems, spectroscopic detection followed by linear analysis allowed spectral discrimination of collagen autofluorescence (M(C)) and the subdermal green fluorescence (M(G)) due to either GFP or the microsphere. Contrast was defined as M(G)/(M(G)+M(C)). The in vivo contrast for GFP using optical fiber and confocal measurements was 0.16 and 0.92, respectively. The ex vivo contrast for a fluorescent microsphere using a confocal system without and with a pinhole was 0.13 and 0.48, respectively. The study demonstrates that a topical optical fiber measurement is affected by collagen autofluorescence, while a confocal microscope can detect subdermal fluorescence while rejecting collagen autofluorescence.
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http://dx.doi.org/10.1117/1.1645798DOI Listing
June 2004

Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia.

Proc Natl Acad Sci U S A 2004 Jan 29;101(2):609-14. Epub 2003 Dec 29.

Research Center, Shriners Hospital for Children, and Departmentof Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239, USA.

Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal degradation and termination of receptor signaling. The defect allows diversion of actively signaling receptors from lysosomes to a recycling pathway where their survival is prolonged, and, as a result, their signaling capacity is increased. The lysosomal targeting defect is additive to other mechanisms proposed to explain the pathogenesis of ACH.
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http://dx.doi.org/10.1073/pnas.2237184100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC327195PMC
January 2004