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1.  miR-218 regulates focal adhesion kinase–dependent TGFβ signaling in fibroblasts 
Molecular Biology of the Cell  2014;25(7):1151-1158.
Unlike other tissues in the body, the oral cavity does not scar. Myofibroblasts are the essential cell type in scar tissue formation; gingival fibroblasts are relatively unable to differentiate into myofibroblasts in response to TGFβ. This differential response is attributed to the relative lack of miR-218 in gingival fibroblasts.
Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)–expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor β (TGFβ) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre–miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFβ to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFβ to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFβ to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.
PMCID: PMC3967977  PMID: 24501422
2.  LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias[S] 
Journal of Lipid Research  2014;55(4):765-772.
We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.
PMCID: PMC3966710  PMID: 24503134
next generation sequencing; DNA diagnosis; familial dyslipidemia; Sanger sequencing; mutations; genetic risk score; polygenic dyslipidemia
3.  Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation 
Arthritis and Rheumatism  2012;64(5):1653-1664.
Enhanced adhesive signaling including activation of the focal adhesion kinase (FAK) is a hallmark of fibroblasts from lung fibrosis patients, and FAK has been therefore hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro.
FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor, PF-562,271, or with siRNA-mediated silencing of FAK, were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and the lungs were harvested for histological and biochemical analysis. Using endothelin-1 (ET-1) as stimulus, cell adhesion and contraction, as well as profibrotic gene expression were studied in fibroblasts isolated from wild type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild type and integrin β1-deficient fibroblasts.
Increased FAK expression and activity are upregulated in fibroblast foci and remodeled vessels in lung fibrosis patients. Pharmacological or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by Jun N-terminal kinase activation through integrin β1/FAK signaling.
These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.
PMCID: PMC3338902  PMID: 22492165
4.  RanBPM expression regulates transcriptional pathways involved in development and tumorigenesis 
RanBPM is a ubiquitous protein that has been reported to regulate several cellular processes through interactions with various proteins. However, it is not known whether RanBPM may regulate gene expression patterns. As it has been shown that RanBPM interacts with a number of transcription factors, we hypothesized that it may have wide ranging effects on gene expression that may explain its function. To test this hypothesis, we generated stable RanBPM shRNA cell lines to analyze the effect of RanBPM on global gene expression. Microarray analyses were conducted comparing the gene expression profile of Hela and HCT116 RanBPM shRNA cells versus control shRNA cells. We identified 167 annotated genes significantly up- or down-regulated in the two cell lines. Analysis of the gene set revealed that down-regulation of RanBPM led to gene expression changes that affect regulation of cell, tissue, and organ development and morphology, as well as biological processes implicated in tumorigenesis. Analysis of Transcription Factor Binding Sites (TFBS) present in the gene set identified several significantly over-represented transcription factors of the Forkhead, HMG, and Homeodomain families of transcription factors, which have previously been demonstrated as having important roles in development and tumorigenesis. In addition, the combined results of these analyses suggested that several signaling pathways were affected by RanBPM down-regulation, including ERK1/2, Wnt, Notch, and PI3K/Akt pathways. Lastly, analysis of selected target genes by quantitative RT-qPCR confirmed the changes revealed by microarray. Several of the genes up-regulated in RanBPM shRNA cells encode proteins with known oncogenic functions, such as the RON tyrosine kinase, the adhesion molecule L1CAM, and transcription factor ELF3/ESE-1, suggesting that RanBPM functions as a tumor suppressor to prevent deregulated expression of these genes. Altogether, these results suggest that RanBPM does indeed function to regulate many genomic events that regulate embryonic, tissue, and cellular development as well as those involved in cancer development and progression.
PMCID: PMC3433104  PMID: 22957307
RanBPM; ERK; Wnt; Notch; microarray; cancer; development
5.  An Analysis of the Myocardial Transcriptome in a Mouse Model of Cardiac Dysfunction with Decreased Cholinergic Neurotransmission 
PLoS ONE  2012;7(6):e39997.
Autonomic dysfunction is observed in many cardiovascular diseases and contributes to cardiac remodeling and heart disease. We previously reported that a decrease in the expression levels of the vesicular acetylcholine transporter (VAChT) in genetically-modified homozygous mice (VAChT KDHOM) leads to decreased cholinergic tone, autonomic imbalance and a phenotype resembling cardiac dysfunction. In order to further understand the molecular changes resulting from chronic long-term decrease in parasympathetic tone, we undertook a transcriptome-based, microarray-driven approach to analyze gene expression changes in ventricular tissue from VAChT KDHOM mice. We demonstrate that a decrease in cholinergic tone is associated with alterations in gene expression in mutant hearts, which might contribute to increased ROS levels observed in these cardiomyocytes. In contrast, in another model of cardiac remodeling and autonomic imbalance, induced through chronic isoproterenol treatment to increase sympathetic drive, these genes did not appear to be altered in a pattern similar to that observed in VAChT KDHOM hearts. These data suggest the importance of maintaining a fine balance between the two branches of the autonomic nervous system and the significance of absolute levels of cholinergic tone in proper cardiac function.
PMCID: PMC3386908  PMID: 22768193
6.  Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling 
PLoS ONE  2012;7(5):e36704.
Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.
PMCID: PMC3344928  PMID: 22574216
7.  Complex learning and information processing by pigeons: a critical analysis 
Three models of conditional discrimination learning by pigeons are described: stimulus configuration learning, the multiple-rule model, and concept learning. A review of the literature reveals that true concept learning is not characteristic of the behavior of pigeons in matching-to-sample, oddity-from-sample, or symbolic matching studies. Instead, pigeons learn a set of sample-specific SD rules. Transfer of the discrimination to novel stimuli, at least along the hue dimension, is predicted by a “coding hypothesis”, which holds that pigeons make a unique, but usually unobserved response, R1, to each sample, and that the comparison stimulus chosen depends on which R1 was emitted in the presence of the sample. Convincing evidence is found that pigeons do code sample hues, but there is little evidence that allows one to infer that the “coding event” must have behavioral properties. Parameters of the conditional discrimination paradigm are identified, and it is shown that by appropriate parametric manipulation, a variety of analogous tasks may be generated for both human and animal subjects. The tasks make possible the comparative study of complex learning, attention, memory, and information processing, with the added advantage that behavior processes may be compared systematically across tasks.
PMCID: PMC1332854  PMID: 16812079
conditional discrimination; matching-to-sample; oddity-from-sample; symbolic matching; coding hypothesis; information processing; short-term memory; attention; rule learning; concept learning; stimulus configuration learning; pigeon
8.  Gingival Fibroblasts Display Reduced Adhesion and Spreading on Extracellular Matrix: A Possible Basis for Scarless Tissue Repair? 
PLoS ONE  2011;6(11):e27097.
Unlike skin, oral gingiva do not scar in response to injury. The basis of this difference is likely to be revealed by comparing the responses of dermal and gingival fibroblasts to fibrogenic stimuli. Previously, we showed that, compared to dermal fibroblasts, gingival fibroblasts are less responsive to the potent pro-fibrotic cytokine TGFβ, due to a reduced production of endothelin-1 (ET-1). In this report, we show that, compared to dermal fibroblasts, human gingival fibroblasts show reduced expression of pro-adhesive mRNAs and proteins including integrins α2 and α4 and focal adhesion kinase (FAK). Consistent with these observations, gingival fibroblasts are less able to adhere to and spread on both fibronectin and type I collagen. Moreover, the enhanced production of ET-1 mRNA and protein in dermal fibroblasts is reduced by the FAK/src inhibitor PP2. Given our previous observations suggesting that fibrotic fibroblasts display elevated adhesive properties, our data suggest that scarring potential may be based, at least in part, on differences in adhesive properties among fibroblasts resident in connective tissue. Controlling adhesive properties may be of benefit in controlling scarring in response to tissue injury.
PMCID: PMC3206920  PMID: 22073262
9.  Mechanical Tension Increases CCN2/CTGF Expression and Proliferation in Gingival Fibroblasts via a TGFβ-Dependent Mechanism 
PLoS ONE  2011;6(5):e19756.
Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours) using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFβ, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1) mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring); but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFβ by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFβ type I (ALK5) receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFβ, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin.
PMCID: PMC3096639  PMID: 21611193
10.  The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts 
Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFβ and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.
PMCID: PMC2443233  PMID: 18600477
CCN2; Microarray; Fibroblasts
11.  FAK Is Required for TGFβ-induced JNK Phosphorylation in Fibroblasts: Implications for Acquisition of a Matrix-remodeling Phenotype 
Molecular Biology of the Cell  2007;18(6):2169-2178.
Transforming growth factor β (TGFβ) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFβ signals that lead to features of connective tissue remodeling, namely formation of an α-smooth muscle actin (α-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFβ causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFβ induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak−/−, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFβ responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFβ signals leading to the control of α-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFβ on connective tissue remodeling by fibroblasts.
PMCID: PMC1877111  PMID: 17409352

Results 1-11 (11)