In-Situ Hybridization of Tropoelastin mRNA during the Development of the Multilayered Neonatal Rat Aortic Smooth Muscle Cell Culture

Toselli, Paul; Faris, Barbara; Sassoon, David; Jackson, Bruce; Franzblau, Carl

doi:10.1016/s0934-8832(11)80084-3

Cited by 17 publications

(6 citation statements)

References 12 publications

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“…By 72 hours, expression was essentially undetectable. Tropoelastin expression continued to increase in control cultures during this time period, consistent with previously reported findings on Eln mRNA expression [32]. A dose response demonstrated that even the lowest dose of SAA tested (0.5 μM) decreased tropoelastin expression (Fig 2B).…”

Section: Resultssupporting

confidence: 91%

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

et al. 2017

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Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis.

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Section: Resultssupporting

confidence: 91%

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

et al. 2017

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“…Timed matings were performed and embryos were harvested to determine the developmental phenotypes. Fixed embryos were prepared for light and electron microscopic analysis and in situ hybridization as described [10, 14]. CK2 expression and activity were determined by in situ hybridization, immuno-blotting, and kinase assay using the CK2-specific peptide substrate RRREEETEEE (Sigma-Genosys) [15].…”

Section: Methodsmentioning

confidence: 99%

Gene targeting of CK2 catalytic subunits

et al. 2008

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Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects.

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“…For reverse transcription (RT)-PCR, the following primer sets were used: 5Ј-ATCAAGGAAGGCTTTA GCAAATGGG-3Ј and 5Ј-GAACCTCGGATTCACATCGTGAGA-3Ј, amplifying a 159-bp product, for brachyury (50); 5Ј-CGGTGTCCAACACAGATCT G-3Ј and 5Ј-TCTCTCGAGGTGGGTTGAC-3Ј, amplifying a 187-bp product, for ANF (50); 5Ј-TGAGGGAGAGCGCAGGCTCAAG-3Ј and 5Ј-TGCTGTC CACGATGGACGTAAGG-3Ј, amplifying a 361-bp product, for myogenin (50); 5Ј-GCCAAGAAGCGGATAGAAGG-3Ј and 5Ј-TGTGGTTCAGGGCTCAGT C-3Ј, amplifying a 499-bp product, for MLC-2V (28) Histology and in situ hybridization. Histologic and in situ hybridization techniques were previously described (45,51). Briefly, freshly dissected whole embryos were fixed for 2 h with 4.3% glutaraldehyde followed by overnight fixation with 1% osmium tetroxide, dehydrated, and embedded in plastic.…”

Section: Fig 1 Ck2␣ Targeting (A) Ck2␣-targeting Vector (Ii) and Tmentioning

confidence: 99%

The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development

Lou

Domı́nguez

Toselli

et al. 2008

Molecular and Cellular Biology

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175

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Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2␣ and/or CK2␣) and two CK2␤ regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2␤ gene is required for early embryonic development, while the CK2␣ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2␣ gene in the mouse germ line. Embryos lacking CK2␣ have a marked reduction in CK2 activity in spite of the presence of the CK2␣ subunit. CK2␣ ؊/؊ embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2␣ appears to play an essential and uncompensated role in mammalian development.

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In-Situ Hybridization of Tropoelastin mRNA during the Development of the Multilayered Neonatal Rat Aortic Smooth Muscle Cell Culture

Cited by 17 publications

References 12 publications

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

Gene targeting of CK2 catalytic subunits

The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development

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