Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the ∼29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.

CCN2 is induced by transforming growth factor-␤ (TGF␤) in fibroblasts and is overexpressed in connective tissue disease. CCN2 has been proposed to be a downstream mediator of TGF␤ action in fibroblasts; however, the role of CCN2 in regulating this process unclear. By using embryonic fibroblasts isolated from ccn2؊/؊ mice, we showed that CCN2 is required for a subset of responses to TGF␤. Affymetrix genome-wide expression profiling revealed that 942 transcripts were induced by TGF␤ greater than 2-fold in ccn2؉/؉ fibroblasts, of which 345 were not induced in ccn2؊/؊ fibroblasts, including pro-adhesive and matrix remodeling genes. Whereas TGF␤ properly induced a generic Smad3-responsive promoter in ccn2؊/؊ fibroblasts, TGF␤-induced activation of focal adhesion kinase (FAK) and Akt was reduced in ccn2؊/؊ fibroblasts. Emphasizing the importance of FAK and Akt activation in CCN2-dependent transcriptional responses to TGF␤ in fibroblasts, CCN2-dependent transcripts were not induced by TGF␤ in fak؊/؊ fibroblasts and were reduced by wortmannin in wild-type fibroblasts. Akt1 overexpression in ccn2؊/؊ fibroblasts rescued the TGF␤-induced transcription of CCN2-dependent mRNA. Finally, induction of TGF␤-induced fibroblast adhesion to fibronectin and type I collagen was significantly diminished in ccn2؊/؊ fibroblasts. Thus in embryonic fibroblasts, CCN2 is a necessary cofactor required for TGF␤ to activate the adhesive FAK/Akt/phosphatidylinositol 3-kinase cascade, FAK/Akt-dependent genes, and adhesion to matrix.Growth factors intimately contribute to the normal wound healing process, regulating chemotaxis, cell proliferation, neovascularization, and extracellular matrix (ECM) 3 synthesis. CCN2 (connective tissue growth factor), a member of the CCN family of proteins, contains 38 conserved cysteine-rich residues and a heparin-binding domain and is chemotactic and mitogenic for connective tissue cells (1-4). However, the physiological role of CCN2 is largely unknown.As an initial approach to elucidate the physiological function of CCN2, mice deleted for the ccn2 gene were recently generated (5). Mice homozygous for a deletion of the ccn2 gene die soon after birth, displaying an inability of the rib cage to ossify properly (5). The phenotype of these mice is consistent with a role for CCN2 in matrix synthesis and remodeling as ccn2Ϫ/Ϫ embryos show reduction in the expression of bone-specific matrix proteins, such as aggrecan (5). We recently found that embryonic fibroblasts isolated from ccn2Ϫ/Ϫ mice showed reduced basal adhesive signaling, including a reduction of FAK and ERK phosphorylation and delays in ␣-smooth muscle actin (␣-SMA) stress fiber formation (6), suggesting that CCN2 plays a key role in mediating the formation of attachments between the cell and matrix at focal adhesions.Although CCN2 was discovered over a decade ago, the precise biological function of CCN2 has remained elusive. CCN2 is expressed in mesenchymal cells in development, is induced during wound healing (4,8), and is overexpressed in fibrosis (7-11)....

Objective 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. Methods 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. Results 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. Conclusion These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

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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.

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.

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We have investigated the effects of chronically elevated glucose concentrations on the pancreatic alpha-cell line alphaTC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature alpha-cell markers glucagon and brn-4 and no induction of the immature alpha-cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the alpha-cell.