Publications by authors named "Timothy J Mead"

20 Publications

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ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

J Hepatol 2021 Apr 13;74(4):893-906. Epub 2020 Nov 13.

Aix-Marseille Univ, CNRS, Developmental Biology Institute of Marseille (IBDM), UMR7288, Parc Scientifique de Luminy, Marseille, France. Electronic address:

Background & Aims: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC).

Methods: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR.

Results: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRβ, IGF1Rβ, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib.

Conclusions: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC.

Lay Summary: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.
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http://dx.doi.org/10.1016/j.jhep.2020.11.008DOI Listing
April 2021

The secreted protease Adamts18 links hormone action to activation of the mammary stem cell niche.

Nat Commun 2020 03 26;11(1):1571. Epub 2020 Mar 26.

Ecole Polytechnique Fédérale de Lausanne, Station 19, CH-1015, Lausanne, Switzerland.

Estrogens and progesterone control breast development and carcinogenesis via their cognate receptors expressed in a subset of luminal cells in the mammary epithelium. How they control the extracellular matrix, important to breast physiology and tumorigenesis, remains unclear. Here we report that both hormones induce the secreted protease Adamts18 in myoepithelial cells by controlling Wnt4 expression with consequent paracrine canonical Wnt signaling activation. Adamts18 is required for stem cell activation, has multiple binding partners in the basement membrane and interacts genetically with the basal membrane-specific proteoglycan, Col18a1, pointing to the basement membrane as part of the stem cell niche. In vitro, ADAMTS18 cleaves fibronectin; in vivo, Adamts18 deletion causes increased collagen deposition during puberty, which results in impaired Hippo signaling and reduced Fgfr2 expression both of which control stem cell function. Thus, Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation.
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http://dx.doi.org/10.1038/s41467-020-15357-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099066PMC
March 2020

Visualization and Quantification of Pericellular Matrix.

Methods Mol Biol 2020 ;2043:261-264

Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.

The pericellular matrix (PCM), also known as the pericellular coat or glycocalyx, lies between the plasma membrane and the interstitial extracellular matrix (ECM). It can have a dramatic influence on cell function because of its presence at the interface between the cell and its microenvironment. A common tool used to demonstrate the PCM is the particle exclusion assay in which fixed red blood cells are utilized to outline the boundary of the cell together with its PCM. PCM visualization and quantification provide opportunities to uncover the roles of ADAMTS proteases in PCM remodeling in many cell types and processes.
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http://dx.doi.org/10.1007/978-1-4939-9698-8_21DOI Listing
April 2020

Alizarin Red and Alcian Blue Preparations to Visualize the Skeleton.

Authors:
Timothy J Mead

Methods Mol Biol 2020 ;2043:207-212

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.

Understanding proteolytic remodeling of extracellular matrix involves the generation of global or conditional knockout mice by homologous recombination in embryonic stem cells or their manipulation through new advanced technologies such as CRISPR-Cas9. These models provide opportunities to understand the roles of ADAMTS genes in skeletogenesis. Whole-mount skeletal preparations are necessary for assessment of the skeletal phenotype. They allow for facile visualization of skeletal patterning, size and shape of skeletal elements, and skeletal structure. This protocol describes the staining of the murine skeleton using Alcian blue to identify cartilage and alizarin red to identify bone.
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http://dx.doi.org/10.1007/978-1-4939-9698-8_17DOI Listing
April 2020

Expression Analysis by RNAscope™ In Situ Hybridization.

Methods Mol Biol 2020 ;2043:173-178

Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.

RNA in situ hybridization has an important place in matrix biology, as the only method that allows for in situ discrimination of precise spatial and temporal patterns of gene expression. Whereas immunohistochemistry shows where a matrix protein localizes, ISH identifies the cell of origin. Thus, these methods provide complementary information for insights on the life cycle of matrix molecules, including ADAMTS proteases. This protocol encompasses the staining of tissue sections to reveal expression of the gene of interest.
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http://dx.doi.org/10.1007/978-1-4939-9698-8_14DOI Listing
April 2020

Adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced fibrillin-2 cleavage.

Matrix Biol 2019 04 7;77:117-128. Epub 2018 Sep 7.

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. Electronic address:

Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10 mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10 mice, similar to Fbn1 mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10 zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10 eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10 mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.
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http://dx.doi.org/10.1016/j.matbio.2018.09.004DOI Listing
April 2019

Exome-chip meta-analysis identifies novel loci associated with cardiac conduction, including ADAMTS6.

Genome Biol 2018 07 17;19(1):87. Epub 2018 Jul 17.

Clinical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK.

Background: Genome-wide association studies conducted on QRS duration, an electrocardiographic measurement associated with heart failure and sudden cardiac death, have led to novel biological insights into cardiac function. However, the variants identified fall predominantly in non-coding regions and their underlying mechanisms remain unclear.

Results: Here, we identify putative functional coding variation associated with changes in the QRS interval duration by combining Illumina HumanExome BeadChip genotype data from 77,898 participants of European ancestry and 7695 of African descent in our discovery cohort, followed by replication in 111,874 individuals of European ancestry from the UK Biobank and deCODE cohorts. We identify ten novel loci, seven within coding regions, including ADAMTS6, significantly associated with QRS duration in gene-based analyses. ADAMTS6 encodes a secreted metalloprotease of currently unknown function. In vitro validation analysis shows that the QRS-associated variants lead to impaired ADAMTS6 secretion and loss-of function analysis in mice demonstrates a previously unappreciated role for ADAMTS6 in connexin 43 gap junction expression, which is essential for myocardial conduction.

Conclusions: Our approach identifies novel coding and non-coding variants underlying ventricular depolarization and provides a possible mechanism for the ADAMTS6-associated conduction changes.
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http://dx.doi.org/10.1186/s13059-018-1457-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048820PMC
July 2018

ADAMTS proteins in human disorders.

Matrix Biol 2018 10 6;71-72:225-239. Epub 2018 Jun 6.

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, United States. Electronic address:

ADAMTS proteins are a superfamily of 26 secreted molecules comprising two related, but distinct families. ADAMTS proteases are zinc metalloendopeptidases, most of whose substrates are extracellular matrix (ECM) components, whereas ADAMTS-like proteins lack a metalloprotease domain, reside in the ECM and have regulatory roles vis-à-vis ECM assembly and/or ADAMTS activity. Evolutionary conservation and expansion of ADAMTS proteins in mammals is suggestive of crucial embryologic or physiological roles in humans. Indeed, Mendelian disorders or birth defects resulting from naturally occurring ADAMTS2, ADAMTS3, ADAMTS10, ADAMTS13, ADAMTS17, ADAMTS20, ADAMTSL2 and ADAMTSL4 mutations as well as numerous phenotypes identified in genetically engineered mice have revealed ADAMTS participation in major biological pathways. Important roles have been identified in a few acquired conditions. ADAMTS5 is unequivocally implicated in pathogenesis of osteoarthritis via degradation of aggrecan, a major structural proteoglycan in cartilage. ADAMTS7 is strongly associated with coronary artery disease and promotes atherosclerosis. Autoantibodies to ADAMTS13 lead to a platelet coagulopathy, thrombotic thrombocytopenic purpura, which is similar to that resulting from ADAMTS13 mutations. ADAMTS proteins have numerous potential connections to other human disorders that were identified by genome-wide association studies. Here, we review inherited and acquired human disorders in which ADAMTS proteins participate, and discuss progress and prospects in therapeutics.
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http://dx.doi.org/10.1016/j.matbio.2018.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146047PMC
October 2018

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation.

Cell Rep 2018 Apr;23(2):485-498

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USA. Electronic address:

Focal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.
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http://dx.doi.org/10.1016/j.celrep.2018.03.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987776PMC
April 2018

The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification.

JCI Insight 2018 04 5;3(7). Epub 2018 Apr 5.

Department of Biomedical Engineering and the Orthopaedic and Rheumatologic Institute, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.

Heterotopic ossification (HO) is a significant clinical problem with incompletely resolved mechanisms. Here, the secreted metalloproteinases ADAMTS7 and ADAMTS12 are shown to comprise a unique proteoglycan class that protects against a tendency toward HO in mouse hindlimb tendons, menisci, and ligaments. Adamts7 and Adamts12 mRNAs were sparsely expressed in murine forelimbs but strongly coexpressed in hindlimb tendons, skeletal muscle, ligaments, and meniscal fibrocartilage. Adamts7-/- Adamts12-/- mice, but not corresponding single-gene mutants, which demonstrated compensatory upregulation of the intact homolog mRNA, developed progressive HO in these tissues after 4 months of age. Adamts7-/- Adamts12-/- tendons had abnormal collagen fibrils, accompanied by reduced levels of the small leucine-rich proteoglycans (SLRPs) biglycan, fibromodulin, and decorin, which regulate collagen fibrillogenesis. Bgn-/0 Fmod-/- mice are known to have a strikingly similar hindlimb HO to that of Adamts7-/- Adamts12-/- mice, implicating fibromodulin and biglycan reduction as a likely mechanism underlying HO in Adamts7-/- Adamts12-/- mice. Interestingly, degenerated human biceps tendons had reduced ADAMTS7 mRNA compared with healthy biceps tendons, which expressed both ADAMTS7 and ADAMTS12. These results suggest that ADAMTS7 and ADAMTS12 drive an innate pathway protective against hindlimb HO in mice and may be essential for human tendon health.
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http://dx.doi.org/10.1172/jci.insight.92941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928868PMC
April 2018

Massive aggrecan and versican accumulation in thoracic aortic aneurysm and dissection.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.

Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications. Thus, aggrecan and versican accumulation in ascending TAAD occurs via increased synthesis and/or reduced proteolytic turnover, and correlates with aortic dissection/rupture in Fbn1mgR/mgR mice. Tissue swelling imposed by aggrecan and versican is proposed to be profoundly deleterious to aortic wall mechanics and smooth muscle cell homeostasis, predisposing to type-A dissections. These proteoglycans provide potential biomarkers for refined risk stratification and timing of elective aortic aneurysm repair.
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http://dx.doi.org/10.1172/jci.insight.97167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922288PMC
March 2018

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma.

J Clin Endocrinol Metab 2017 05;102(5):1631-1641

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195.

Context: Leiomyomas have abundant extracellular matrix (ECM), with upregulation of versican, a large proteoglycan.

Objective: We investigated ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) protease-mediated versican cleavage in myometrium and leiomyoma and the effect of versican knockdown in leiomyoma cells.

Design: We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and RNA in situ hybridization for analysis of myometrium, leiomyoma and immortalized myometrium and leiomyoma cells. Short interfering RNA (siRNA) was used to knockdown versican in leiomyoma cells.

Setting: This study was performed in an academic laboratory.

Patients: Study subjects were women with symptomatic or asymptomatic leiomyoma.

Main Outcome Measures: We quantified messenger RNAs (mRNAs) for versican splice variants. We identified ADAMTS-cleaved versican in myometrium and leiomyoma and ADAMTS messenger RNAs and examined the effect of VCAN siRNA on smooth muscle differentiation and expression of estrogen and progesterone receptors.

Results: The women in the symptomatic group (n = 7) had larger leiomyoma (P = 0.01), heavy menstrual bleeding (P < 0.01), and lower hemoglobin levels (P = 0.02) compared with the asymptomatic group (n = 7), but were similar in age and menopausal status. Versican V0 and V1 isoforms were upregulated in the leiomyomas of symptomatic versus asymptomatic women (P = 0.03 and P = 0.04, respectively). Abundant cleaved versican was detected in leiomyoma and myometrium, as well as in myometrial and leiomyoma cell lines. ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) were upregulated in symptomatic leiomyomas. VCAN siRNA did not effect cell proliferation, apoptosis, or smooth muscle markers, but reduced ESR1 and PR-A expression (P = 0.001 and P = 0.002, respectively).

Conclusions: Versican in myometrium, leiomyomas and in the corresponding immortalized cells is cleaved by ADAMTS proteases. VCAN siRNA suppresses production of estrogen receptor 1 and progesterone receptor-A. These findings have implications for leiomyoma growth.
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http://dx.doi.org/10.1210/jc.2016-3527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443325PMC
May 2017

The SOX9 upstream region prone to chromosomal aberrations causing campomelic dysplasia contains multiple cartilage enhancers.

Nucleic Acids Res 2015 Jun 4;43(11):5394-408. Epub 2015 May 4.

Department of Cellular & Molecular Medicine, and Orthopaedic and Rheumatologic Research Center, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USA

Two decades after the discovery that heterozygous mutations within and around SOX9 cause campomelic dysplasia, a generalized skeleton malformation syndrome, it is well established that SOX9 is a master transcription factor in chondrocytes. In contrast, the mechanisms whereby translocations in the --350/-50-kb region 5' of SOX9 cause severe disease and whereby SOX9 expression is specified in chondrocytes remain scarcely known. We here screen this upstream region and uncover multiple enhancers that activate Sox9-promoter transgenes in the SOX9 expression domain. Three of them are primarily active in chondrocytes. E250 (located at -250 kb) confines its activity to condensed prechondrocytes, E195 mainly targets proliferating chondrocytes, and E84 is potent in all differentiated chondrocytes. E84 and E195 synergize with E70, previously shown to be active in most Sox9-expressing somatic tissues, including cartilage. While SOX9 protein powerfully activates E70, it does not control E250. It requires its SOX5/SOX6 chondrogenic partners to robustly activate E195 and additional factors to activate E84. Altogether, these results indicate that SOX9 expression in chondrocytes relies on widely spread transcriptional modules whose synergistic and overlapping activities are driven by SOX9, SOX5/SOX6 and other factors. They help elucidate mechanisms underlying campomelic dysplasia and will likely help uncover other disease mechanisms.
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http://dx.doi.org/10.1093/nar/gkv426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477657PMC
June 2015

Proliferation assays (BrdU and EdU) on skeletal tissue sections.

Methods Mol Biol 2014 ;1130:233-243

Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.

Assessing cell proliferation in situ is an important phenotyping component of skeletal tissues from development to adult stages and disease. Various methods exist including immunostaining for proteins and protein modifications associated with specific steps of the cell cycle, but the gold standard is to quantify the percentage of DNA-synthesizing cells. The thymidine analog 5-bromo-2'-deoxyuridine (BrdU) has been widely used in the last decades for this purpose, with the inconvenience that its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to nucleosides in genomic DNA. In 2008, Salic and Mitchison developed a new method and proved it to be quicker, simpler, and highly sensitive in non-skeletal tissues. This method relies on incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into de novo DNA. This other thymidine analog is readily detected by click chemistry, i.e., covalent cross-linking of its ethynyl group with a fluorescent azide, a molecule small enough to diffuse freely through native tissues and DNA. Here, we describe and compare the BrdU and EdU approaches in skeletal tissues and conclude that in these tissues too EdU provides an easy and very sensitive alternative to BrdU.
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http://dx.doi.org/10.1007/978-1-62703-989-5_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074019PMC
September 2014

A far-upstream (-70 kb) enhancer mediates Sox9 auto-regulation in somatic tissues during development and adult regeneration.

Nucleic Acids Res 2013 Apr 28;41(8):4459-69. Epub 2013 Feb 28.

Department of Cellular and Molecular Medicine, and Orthopaedic and Rheumatologic Research Center, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USA.

SOX9 encodes a transcription factor that presides over the specification and differentiation of numerous progenitor and differentiated cell types, and although SOX9 haploinsufficiency and overexpression cause severe diseases in humans, including campomelic dysplasia, sex reversal and cancer, the mechanisms underlying SOX9 transcription remain largely unsolved. We identify here an evolutionarily conserved enhancer located 70-kb upstream of mouse Sox9 and call it SOM because it specifically activates a Sox9 promoter reporter in most Sox9-expressing somatic tissues in transgenic mice. Moreover, SOM-null fetuses and pups reduce Sox9 expression by 18-37% in the pancreas, lung, kidney, salivary gland, gut and liver. Weanlings exhibit half-size pancreatic islets and underproduce insulin and glucagon, and adults slowly recover from acute pancreatitis due to a 2-fold impairment in Sox9 upregulation. Molecular and genetic experiments reveal that Sox9 protein dimers bind to multiple recognition sites in the SOM sequence and are thereby both necessary and sufficient for enhancer activity. These findings thus uncover that Sox9 directly enhances its functions in somatic tissue development and adult regeneration through SOM-mediated positive auto-regulation. They provide thereby novel insights on molecular mechanisms controlling developmental and disease processes and suggest new strategies to improve disease treatments.
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http://dx.doi.org/10.1093/nar/gkt140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632127PMC
April 2013

Notch signaling and the developing skeleton.

Adv Exp Med Biol 2012 ;727:114-30

Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Notch signaling is an important regulator of skeletogenesis at multiple developmental stages. The Notch signaling pathway is involved in the promotion of somite segmentation, patterning and differentiation into sclerotome pre-chondrogenic cells to allow for appropriate axial skeleton development. In addition, studies performed in vitro and in vivo demonstrate that Notch signaling suppresses chondrogenic and osteoblastic differentiation and negatively regulates osteoclast formation and proliferation. Through the use of in vitro and in vivo approaches, Notch signaling has been shown to regulate somitogenesis, chondrogenesis, osteoblastogenesis and osteoclastogenesis that ultimately affect skeletogenesis. Dysregulation of Notch signaling results in congenital skeletal malformations that could reveal therapeutic potential.
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http://dx.doi.org/10.1007/978-1-4614-0899-4_9DOI Listing
April 2012

Notch pathway regulation of neural crest cell development in vivo.

Dev Dyn 2012 Feb 3;241(2):376-89. Epub 2012 Jan 3.

The Heart Institute, Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

Background: The function of Notch signaling in murine neural crest-derived cell lineages in vivo was examined.

Results: Conditional gain (Wnt1Cre;Rosa(Notch)) or loss (Wnt1Cre;RBP-J(f/f)) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa(Notch) embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP-J(f/f) mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa(Notch) and Wnt1Cre;RBP-J(f/f) embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa(Notch) embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP-J(f/f) embryos.

Conclusions: Together, these studies demonstrate essential cell-autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease.
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http://dx.doi.org/10.1002/dvdy.23717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266628PMC
February 2012

Requirements for Jag1-Rbpj mediated Notch signaling during early mouse lens development.

Dev Dyn 2012 Mar 25;241(3):493-504. Epub 2012 Jan 25.

Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.

Background: During vertebrate lens development, the lens placode in the embryonic ectoderm invaginates into a lens vesicle, which then separates from the surface epithelium, followed by two waves of fiber cell differentiation. In the mouse, multiple labs have shown that Jag1-Notch signaling is critically required during the second wave of lens fiber cell formation. However, Notch signaling appears to play no obvious role during lens induction or morphogenesis, although multiple pathway genes are expressed at these earlier stages.

Results: Here, we explored functions for Notch signaling specifically during early lens development, by using the early-acting AP2α-Cre driver to delete Jag1 or Rbpj. We found that Jag1 and Rbpj are not required during lens induction, but are necessary for proper lens vesicle separation from the surface ectoderm.

Conclusions: We conclude that precise levels of Notch signaling are essential during lens vesicle morphogenesis. In addition, AP2α-Cre-mediated deletion of Rbpj resulted in embryos with cardiac outflow tract and liver deformities, and perinatal lethality.
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http://dx.doi.org/10.1002/dvdy.23739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288604PMC
March 2012

Notch pathway regulation of chondrocyte differentiation and proliferation during appendicular and axial skeleton development.

Proc Natl Acad Sci U S A 2009 Aug 9;106(34):14420-5. Epub 2009 Jul 9.

Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

The role of Notch signaling in cartilage differentiation and maturation in vivo was examined. Conditional Notch pathway gain and loss of function was achieved using a Cre/loxP approach to manipulate Notch signaling in cartilage precursors and chondrocytes of the developing mouse embryo. Conditional overexpression of activated Notch intracellular domain (NICD) in the chondrocyte lineage results in skeletal malformations with decreased cartilage precursor proliferation and inhibited hypertrophic chondrocyte differentiation. Likewise, expression of NICD in cartilage precursors inhibits sclerotome differentiation, resulting in severe axial skeleton abnormalities. Furthermore, conditional loss of Notch signaling via RBP-J gene deletion in the chondrocyte lineage results in increased chondrocyte proliferation and skeletal malformations consistent with the observed increase in hypertrophic chondrocytes. In addition, the Notch pathway inhibits expression of Sox9 and its target genes required for normal chondrogenic cell proliferation and differentiation. Together, our results demonstrate that appropriate Notch pathway signaling is essential for proper chondrocyte progenitor proliferation and for the normal progression of hypertrophic chondrocyte differentiation into bone in the developing appendicular and axial skeletal elements.
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http://dx.doi.org/10.1073/pnas.0902306106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732875PMC
August 2009

Lymphangiogenesis in the developing lung promoted by VEGF-A.

Microvasc Res 2006 Jul-Sep;72(1-2):62-73. Epub 2006 Jun 23.

Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

Understanding the basic processes of late-stage pulmonary vascular development is essential as this period corresponds to the stage when preterm infants have increased chance of survival. During this period, refinement of the gas exchange unit leads to close apposition of the capillary vasculature and airway epithelium through thinning of the mesenchyme, formation of alveolar septae and functional adaptation of endothelial cells into vessels including pulmonary lymphatics. The pulmonary lymphatic network promotes efficient gas exchange through maintaining interstitial fluid balance. Through conditional transgene regulation, we found that a modest, pathologically relevant increase in vascular endothelial growth factor A (VEGF-A) in distal lung during only the perinatal period adversely affected final refinement of the gas exchange unit. VEGF-A induction disrupted the established vascular network, increased endothelial cell number, altered endothelial ultrastructure and reduced mesenchymal thinning. In addition, VEGF-A induction caused a 3-fold increase in small vessels identified as lymphatics in distal lung. mRNA levels of lymphangiogenic factors VEGF-D/-C were unchanged, while levels of the cognate receptor VEGFR-3 increased. The responses to VEGF-A induction in the perinatal period differ from those during early lung development when endothelial migration, but not proliferation altered initial vascular patterning (Akeson, A.L., Greenberg, J.M., Cameron, J.E., Thompson, F.Y., Brooks, S.K., Wiginton, D., Whitsett, J.A., 2003. Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung. Dev. Biol. 264, 443-455). The late-stage response resembles that of adult lung to VEGF-inducing stimuli including injury and disease. These data suggest that VEGF-A influences the balance between development of blood and lymphatic vasculature during lung organogenesis.
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http://dx.doi.org/10.1016/j.mvr.2006.05.002DOI Listing
January 2007