Medicine and Health Sciences Commons^™

Med25 Limits Master Regulators That Govern Adipogenesis, Jasmine Saunders, Kunal Sikder, Elizabeth Phillips, Anurag Ishwar, David Mothy, Kenneth B. Margulies, Jason C. Choi

Department of Medicine Faculty Papers

Mediator 25 (Med25) is a member of the mediator complex that relays signals from transcription factors to the RNA polymerase II machinery. Multiple transcription factors, particularly those involved in lipid metabolism, utilize the mediator complex, but how Med25 is involved in this context is unclear. We previously identified Med25 in a translatome screen of adult cardiomyocytes (CMs) in a novel cell type-specific model of LMNA cardiomyopathy. In this study, we show that Med25 upregulation is coincident with myocardial lipid accumulation. To ascertain the role of Med25 in lipid accumulation, we utilized iPSC-derived and neonatal CMs to recapitulate the in vivo …

Anxiolytics For Bronchodilation: Refinements To Gaba, Ajay P Nayak, Steven S An

Department of Medicine Faculty Papers

No abstract provided.

D121 Located Within The Dry Motif Of P2y12 Is Essential For P2y12-Mediated Platelet Function., Carol Dangelmaier, Benjamin Mauri, Akruti Patel, Satya P Kunapuli, John C Kostyak

Department of Medicine Faculty Papers

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. …

Grk2 Regulates Adp Signaling In Platelets Via P2y1 And P2y12., Xuefei Zhao, Matthew Cooper, James V Michael, Yanki Yarman, Aiden Baltz, J Kurt Chuprun, Walter J Koch, Steven E. Mckenzie, Maurizio Tomaiuolo, Timothy J Stalker, Li Zhu, Peisong Ma

Department of Medicine Faculty Papers

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid …

Inhibition Of Nadph Oxidase Blocks Netosis And Reduces Thrombosis In Heparin-Induced Thrombocytopenia, Halina H L Leung, Jose Perdomo, Zohra Ahmadi, Feng Yan, Steven E. Mckenzie, Beng H Chong

Department of Medicine Faculty Papers

Heparin-induced thrombocytopenia (HIT) is associated with severe and potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes induced ROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant neutrophil extracellular traps (NETs) and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis …

Neural Crest Cell-Autonomous Roles Of Fibronectin In Cardiovascular Development., Xia Wang, Sophie Astrof

Department of Medicine Faculty Papers

The chemical and mechanical properties of extracellular matrices (ECMs) modulate diverse aspects of cellular fates; however, how regional heterogeneity in ECM composition regulates developmental programs is not well understood. We discovered that fibronectin 1 (Fn1) is expressed in strikingly non-uniform patterns during mouse development, suggesting that regionalized synthesis of the ECM plays cell-specific regulatory roles during embryogenesis. To test this hypothesis, we ablated Fn1 in the neural crest (NC), a population of multi-potent progenitors expressing high levels of Fn1. We found that Fn1 synthesized by the NC mediated morphogenesis of the aortic arch artery and differentiation of NC cells into …

Fibronectin Signals Through Integrin Α5Β1 To Regulate Cardiovascular Development In A Cell Type-Specific Manner., Dongying Chen, Xia Wang, Dong Liang, Julie Gordon, Ashok Mittal, Nancy Manley, Karl Degenhardt, Sophie Astrof

Department of Medicine Faculty Papers

Fibronectin (Fn1) is an evolutionarily conserved extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to mid-gestation lethality from cardiovascular defects. However, severe morphogenetic defects that occur early in embryogenesis in these embryos precluded assigning a direct role for Fn1 in cardiovascular development. We noticed that Fn1 is expressed in strikingly non-uniform patterns during mouse embryogenesis, and that its expression is particularly enriched in the pharyngeal region corresponding with the pharyngeal arches 3, 4, and 6. This region bears a special importance for the developing cardiovascular system, and we hypothesized that the localized enrichment of Fn1 in …

Expression Of Enteropathogenic Escherichia Coli Map Is Significantly Different Than That Of Other Type Iii Secreted Effectors In Vivo., Mai Nguyen, Jason Rizvi, Gail Hecht

Department of Medicine Faculty Papers

The enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement (LEE)-encoded effectors EspF and Map are multifunctional and have an impact on the tight junction barrier while the non-LEE-encoded proteins NleH1 and NleH2 possess significant anti-inflammatory activity. In order to address the temporal expression of these important genes in vivo, their promoters were cloned upstream of the luxCDABE operon, and luciferase expression was measured in EPEC-infected mice by bioluminescence using an in vivo imaging system (IVIS). Bioluminescent images of living mice, of excised whole intestines, and of whole intestines longitudinally opened and washed were assessed. The majority of bioluminescent bacteria localized …

Smooth Muscle Fascicular Reorientation Is Required For Esophageal Morphogenesis And Dependent On Cdo., Anthony I Romer, Jagmohan Singh, Satish Rattan, Robert S Krauss

Department of Medicine Faculty Papers

Postnatal maturation of esophageal musculature involves proximal-to-distal replacement of smooth muscle with skeletal muscle by elusive mechanisms. We report that this process is impaired in mice lacking the cell surface receptor Cdo and identify the underlying developmental mechanism. A myogenic transition zone containing proliferative skeletal muscle precursor cells migrated in a proximal-distal direction, leaving differentiated myofibers in its wake. Distal to the transition zone, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. Consequently, a path was cleared for the transition zone, and smooth muscle ultimately occupied only the …

Human Genome-Wide Association And Mouse Knockout Approaches Identify Platelet Supervillin As An Inhibitor Of Thrombus Formation Under Shear Stress., Leonard C. Edelstein, Elizabeth J. Luna, Ian B. Gibson, Molly Bray, Ying Jin, Altaf Kondkar, Srikanth Nagalla, Nacima Hadjout-Rabi, Tara C. Smith, Daniel Covarrubias, Stephen N. Jones, Firdos Ahmad, Moritz Stolla, Xianguo Kong, Zhiyou Fang, Wolfgang Bergmeier, Chad Shaw, Suzanne M. Leal, Paul Bray

Department of Medicine Faculty Papers

BACKGROUND: High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study and confirmatory experiments in human and animal platelets.

METHODS AND RESULTS: Closure times in the shear-dependent platelet function analyzer (PFA)-100 were measured on healthy, nondiabetic European Americans (n=125) and blacks (n=116). A genome-wide association (P

CONCLUSIONS: We show for the first time that (1) platelets contain supervillin; (2) platelet thrombus formation in the PFA-100 is associated with human SVIL variants and low SVIL expression; and (3) …

Protein Kinase Cδ And C-Abl Kinase Are Required For Transforming Growth Factor Β Induction Of Endothelial-Mesenchymal Transition In Vitro., Zhaodong Li, Sergio A. Jimenez

Department of Medicine Faculty Papers

OBJECTIVE: The origin of the mesenchymal cells responsible for the intimal fibrosis in systemic sclerosis (SSc) has not been fully identified. The present study was undertaken to investigate whether subendothelial mesenchymal cells may emerge through transdifferentiation of endothelial cells (ECs) into myofibroblasts via endothelial-mesenchymal transition (EndoMT) in vitro and to explore the signaling pathways involved in this process.

METHODS: Primary mouse pulmonary ECs isolated by immunomagnetic methods with sequential anti-CD34 and anti-CD102 antibody selection were cultured in monolayers. Cell morphology and diacetylated low-density lipoprotein uptake assays confirmed their EC characteristics. The induction of EndoMT was assessed by determination of α-smooth …

The Interplay Between Nf-Kappab And E2f1 Coordinately Regulates Inflammation And Metabolism In Human Cardiac Cells., Xavier Palomer, David Álvarez-Guardia, Mercy M Davidson, Tung O Chan, Arthur M Feldman, Manuel Vázquez-Carrera

Department of Medicine Faculty Papers

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, …

Reduction Of Sympathetic Activity Via Adrenal-Targeted Grk2 Gene Deletion Attenuates Heart Failure Progression And Improves Cardiac Function After Myocardial Infarction., Anastasios Lymperopoulos, Giuseppe Rengo, Erhe Gao, Steven N. Ebert, Gerald W. Dorn, Walter J. Koch

Department of Medicine Faculty Papers

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells …

Decreased Expression Of Caveolin 1 In Patients With Systemic Sclerosis: Crucial Role In The Pathogenesis Of Tissue Fibrosis., Francesco Del Galdo, Federica Sotgia, Cecilia J. De Almeida, Jean-Francois Jasmin, Megan Musick, Michael P. Lisanti, Sergio A. Jimenez

Department of Medicine Faculty Papers

OBJECTIVE: Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro.

METHODS: CAV1 expression in tissues was analyzed by …

Beta3 Integrin Haplotype Influences Gene Regulation And Plasma Von Willebrand Factor Activity, Katie E. Payne, Paul F. Bray, Peter J. Grant, Angela M. Carter

Department of Medicine Faculty Papers

The Leu33Pro polymorphism of the gene encoding beta(3) integrin (ITGB3) is associated with acute coronary syndromes and influences platelet aggregation. Three common promoter polymorphisms have also been identified. The aims of this study were to (1) investigate the influence of the ITGB3 -400C/A, -425A/C and -468G/A promoter polymorphisms on reporter gene expression and nuclear protein binding and (2) determine genotype and haplotype associations with platelet alpha(IIb)beta(3) receptor density. Promoter haplotypes were introduced into an ITGB3 promoter-pGL3 construct by site directed mutagenesis and luciferase reporter gene expression analysed in HEL and HMEC-1 cells. Binding of nuclear proteins was assessed by electrophoretic …

Adiponectin Deficiency Increases Leukocyte-Endothelium Interactions Via Upregulation Of Endothelial Cell Adhesion Molecules In Vivo, Raogo Ouedraogo, Yulan Gong, Brett Berzins, Xiandong Wu, Kalyankar Mahadev, Kelly Hough, Lawrence Chan, Barry J. Goldstein, Rosario Scalia

Department of Medicine Faculty Papers

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad^–/–) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad^–/– mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad …

Characterization Of Hard2, A Processed Hard1 Gene Duplicate, Encoding A Human Protein N-Alpha-Acetyltransferase., Thomas Arnesen, Matthew J Betts, Frédéric Pendino, David A Liberles, Dave Anderson, Jaime Caro, Xianguo Kong, Jan E Varhaug, Johan R Lillehaug

Department of Medicine Faculty Papers

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing epsilon-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal alpha-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. …

Multiple Metabolic Hits Converge On Cd36 As Novel Mediator Of Tubular Epithelial Apoptosis In Diabetic Nephropathy., Katalin Susztak, Emilio Ciccone, Peter Mccue, Kumar Sharma, Erwin P Böttinger

Department of Medicine Faculty Papers

BACKGROUND: Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.

METHODS AND FINDINGS: We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced …

Esophageal Muscle Physiology And Morphogenesis Require Assembly Of A Collagen Xix-Rich Basement Membrane Zone., Hideaki Sumiyoshi, Niv Mor, Sui Y Lee, Stephen Doty, Scott Henderson, Shizuko Tanaka, Hidekatsu Yoshioka, Satish Rattan, Francesco Ramirez

Department of Medicine Faculty Papers

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, …

The Nad(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation Of H202 And Plays An Integral Role In Insulin Signal Transduction, Kalyankar Mahadev, Hiroyuki Motoshima, Xiangdong Wu, Jean Marie Ruddy, Rebecca S. Arnold, Guangjie Cheng, J. David Lambeth, Barry J. Goldstein

Department of Medicine Faculty Papers

Insulin stimulation of target cells elicits a burst of H₂O₂ that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative …

Transcriptional Inhibition Of Type I Collagen Gene Expression In Scleroderma Fibroblasts By The Antineoplastic Drug Ecteinascidin 743., Natalia Louneva, Biagio Saitta, David J Herrick, Sergio A. Jimenez

Department of Medicine Faculty Papers

We previously showed that COL1A1 expression is up-regulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70-90% at 700 pM without affecting cell viability. The same drug concentration caused 60-80% reduction …

Regulation Of Human Col9a1 Gene Expression. Activation Of The Proximal Promoter Region By Sox9., Ping Zhang, Sergio A. Jimenez, David G Stokes

Department of Medicine Faculty Papers

The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter …

A Tandem Duplication Within The Fibrillin 1 Gene Is Associated With The Mouse Tight Skin Mutation., Linda D. Siracusa, Rodney Mcgrath, Qing Ma, John J. Moskow, Jayanthi Manne, Paul J. Christner, Arthur M. Buchberg, Sergio A. Jimenez

Department of Medicine Faculty Papers

Mice carrying the Tight skin (Tsk) mutation have thickened skin and visceral fibrosis resulting from an accumulation of extracellular matrix molecules. These and other connective tissue abnormalities have made Tskl + mice models for scleroderma, hereditary emphysema, and myocardial hypertrophy. Previously we localized Tsk to mouse chromosome 2 in a region syntenic with human chromosome 15. The microfibrillar glycoprotein gene, fibrillin 1 (FBN1), on human chromosome 15q, provided a candidate for the Tsk mutation. We now demonstrate that the Tsk chromosome harbors a 30- to 40-kb genomic duplication within the Fbn1 gene that results in a larger than normal in-frame …

Increased Alpha 1(I) Procollagen Gene Expression In Tight Skin (Tsk) Mice Myocardial Fibroblasts Is Due To A Reduced Interaction Of A Negative Regulatory Sequence With Ap-1 Transcription Factor., Neena Philips, Reza I. Bashey, Sergio A. Jimenez

Department of Medicine Faculty Papers

The TSK mouse, a model of fibrosis, displays exaggerated connective tissue accumulation in skin and visceral organs including the heart. To study the mechanisms of myocardial fibrosis in TSK mice, we established several strains of TSK mice myocardial fibroblasts in culture and examined the regulation of collagen gene expression in these cells. These strains displayed increased collagen gene expression in comparison with myocardial fibroblasts established from normal mice. On an average, the TSK myocardial fibroblast cultures showed a 4-fold increase in collagen synthesis and 4.4- and 3.6-fold increases, respectively, in alpha 1(I) and alpha 1(III) collagen mRNA steady state levels. …

Epidermal Growth Factor Coordinately Regulates The Expression Of Prostaglandin G/H Synthase And Cytosolic Phospholipase A2 Genes In Embryonic Mouse Cells., Kenneth P. Chepenik, Arturo Diaz, Sergio A. Jimenez

Department of Medicine Faculty Papers

Confluent, primary cultures of mouse embryo palate mesenchyme (MEPM) cells are refractory to activation of phospholipase A2 (PLA2) by the calcium ionophore A23187. However, treatment of these cultures with epidermal growth factor (EGF) permits the cells to activate PLA2 in response to A23187. We have developed this finding by exploring molecular mechanisms by which growth factors modulate mobilization and metabolism of arachidonic acid. We found chronic treatment (> 6 h) of confluent MEPM cells with EGF (a) increases their ability to metabolize exogenous arachidonic acid to prostaglandin E2 (PGE2) and (b) stimulated constitutive expression of activities of PLA2 and cyclooxygenase …

Functional Analysis Of Human Alpha 1(I) Procollagen Gene Promoter. Differential Activity In Collagen-Producing And -Nonproducing Cells And Response To Transforming Growth Factor Beta 1., Sergio A. Jimenez, John A. Varga, Anne Olsen, Liye Li, Arturo Diaz, Janet Herhal, Julie Koch

Department of Medicine Faculty Papers

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5'-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5' end points from -804 to -174 bp. A further 5' deletion to -84 bp caused a marked reduction …

Expression Of A Human Cartilage Procollagen Gene (Col2a1) In Mouse 3t3 Cells., Leena Ala-Kokko, James Hyland, Carol Smith, Kari I. Kivirikko, Sergio A. Jimenez, Darwin J. Prockop

Department of Medicine Faculty Papers

Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational enzymes. Here, two DNA constructs were designed so that the promoter region of the gene for the pro-alpha 1(I) chain of human type I procollagen drove expression of the human type II procollagen gene in mouse NIH 3T3 cells, a culture line that normally synthesizes type I procollagen but not any cartilage-specific protein such as type II procollagen. Both constructs were expressed as both mRNA and protein. In clones expressing the construct at high levels, …