Transgene Delivery to Cultured Keratinocytes via Replication-Deficient Adenovirus Vectors

Ramirez, Vincent P.; Aneskievich, Brian J.

doi:10.1007/7651_2013_43

Cited by 3 publications

(2 citation statements)

References 17 publications

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“…The cells were plated on 6- or 24-well plates at a density of 6.8 × 10 5 or 1.5 × 10 5 cells per well, respectively. Twenty-four hours after plating, cells were infected with an adenovirus construct expressing TNIP1 (Ad-TNIP1) or LacZ as a control (Ad-LacZ) at a multiplicity of infection (MOI) of 500 using 8 μg/ml Polybrene (Millipore, Billerica, MA) [18] in DMEM/F12 media containing 2% FBS. Sixteen hours post-infection, the viral mixture was aspirated and media replaced.…”

Section: Methodsmentioning

confidence: 99%

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TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors

Ramirez

Krueger

Aneskievich

2015

Gene

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TNFα-induced protein 3-interacting protein 1 (TNIP1) represses signaling pathways initiated by specific nuclear and transmembrane receptors. This effect results in reduced activity of distinct transcription factors such as retinoic acid receptors (RAR), peroxisome-proliferator-activated receptors (PPAR), and NFκB. TNIP1-null and TNIP1-knockin defective for ubiquitin-binding mice show increased liver apoptosis, and enlarged spleen and lymph nodes, respectively. To complement current knowledge of TNIP1’s broad physiologic functions as interpreted from in vivo studies and specific expression consequences from transcription factor repression, we determined effects of excess TNIP1 on global gene regulation. Following experimentally increased expression of TNIP1 in cultured keratinocytes, our gene expression microarray analysis not only confirmed TNIP1’s association in previously known pathways and functions but also found a novel TNIP1-regulated pathway – the cell stress response. Under standard culture conditions, expression of several heat shock proteins, including HSPA1A, HSPA6, DNAJA1 and DNAJB1, was reduced. In heat-stressed conditions, differential regulation of HSPA1A and HSPA6 was observed, where only HSPA6 expression was reduced after heat-shock. Using HSPA6 as a model to elucidate the mechanism of the TNIP1-mediated HSP repression, we determined TNIP1 likely represses HSPs through factors other than RAR, PPAR or NFκB despite presence of these factors’ binding sites in the HSPA6 promoter. These results indicate that regulation of HSPs may be through a yet unknown TNIP1-associated pathway. Additionally, these results suggest TNIP1’s reduction of HSP expression levels could negatively impact HSP chaperone capacity or their participation in the cell stress response.

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Section: Methodsmentioning

confidence: 99%

“…Twenty-four hours after plating, cells were infected with Ad-TNIP1 or Ad-LacZ at a MOI of 500 using Polybrene in DMEM/F12 media supplemented with 2% FBS [18]. Eight hours later, the appropriate HSPA6 promoter pGL4.10 plasmid (200 ng) and pCMV-β galactosidase (100 ng) was transfected using Fugene6 (Promega) using 100 μL serum-free media.…”

Section: Methodsmentioning

confidence: 99%

TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors

Ramirez

Krueger

Aneskievich

2015

Gene

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Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions

Ramirez

Stamatis

Shmukler

et al. 2015

Cell Stress and Chaperones

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Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B′) is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced. Despite this response in other cells, little is known about its control in keratinocytes. We examined endogenous human keratinocyte HSPA6 expression and localized some responsible transcription factor sites in a cloned HSPA6 3 kb promoter. Using promoter 5′ truncations and deletions, negative and positive regulatory regions were found throughout the 3 kb promoter. A region between −346 and −217 bp was found to be crucial to HSPA6 basal expression and stress inducibility. Site-specific mutations and DNA-binding studies show that a previously uncharacterized AP1 site contributes to the basal expression and maximal stress induction of HSPA6. Additionally, a new heat shock element (HSE) within this region was defined. While this element mediates increased transcriptional response in thermally stressed HaCaT keratinocytes, it preferentially binds a stress-inducible factor other than heat shock factor (HSF)1 or HSF2. Intriguingly, this newly characterized HSPA6 HSE competes HSF1 binding a consensus HSE and binds both HSF1 and HSF2 from other epithelial cells. Taken together, our results demonstrate that the HSPA6 promoter contains essential negative and positive promoter regions and newly identified transcription factor targets, which are key to the basal and stress-inducible expression of HSPA6. Furthermore, these results suggest that an HSF-like factor may preferentially bind this newly identified HSPA6 HSE in HaCaT cells.

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Cationic polymers for enhancing CpG oligodeoxynucleotides-mediated cancer immunotherapy

Cai

Ling

et al. 2019

European Polymer Journal

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Transgene Delivery to Cultured Keratinocytes via Replication-Deficient Adenovirus Vectors

Cited by 3 publications

References 17 publications

TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors

TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors

Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions

Cationic polymers for enhancing CpG oligodeoxynucleotides-mediated cancer immunotherapy

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