Exome sequencing identifies novel mutation signatures of UV radiation and trichostatin A in primary human keratinocytes

Edelweiss (Leontopodium alpinumCass.) is traditionally employed in folk medicine as an anti-inflammatory remedy. In nature, the plant is sparsely available and protected; therefore production of callus cultures was established. A concentrated ethanolic extract of culture homogenate, with leontopodic acid representing55±2% of the total phenolic fraction (ECC55), was characterized for anti-inflammatory properties in primary human keratinocytes (PHKs) and endotheliocytes (HUVECs). Inflammatory responses were induced by UVA+UVB, lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL), and a mixture of proinflammatory cytokines. Trichostatin A, a sirtuin inhibitor, was used to induce keratinocyte inflammatory senescence. ECC55 (10–50 μg/mL) protected PHK from solar UV-driven damage, by enhancing early intracellular levels of nitric oxide, although not affecting UV-induced expression of inflammatory genes. Comparison of the dose-dependent inhibition of chemokine (IL-8, IP-10, MCP-1) and growth factor (GM-CSF) release from PHK activated by TNFα+ IFNγshowed that leontopodic acid was mainly responsible for the inhibitory effects of ECC55. Sirtuin-inhibited cell cycle, proliferation, and apoptosis markers were restored by ECC55. The extract inhibited LPS-induced IL-6 and VCAM1 genes in HUVEC, as well as oxLDL-induced selective VCAM1 overexpression.Conclusion.Edelweiss cell cultures could be a valuable source of anti-inflammatory substances potentially applicable for chronic inflammatory skin diseases and bacterial and atherogenic inflammation.

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UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins

Journal of Virology ◽

10.1128/jvi.01123-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 7

Author(s):

Laura Pacini ◽

Maria Grazia Ceraolo ◽

Assunta Venuti ◽

Giusi Melita ◽

Uzma A. Hasan ◽

...

Keyword(s):

Transcription Factors ◽

Uv Radiation ◽

Uv Irradiation ◽

Human Keratinocytes ◽

Skin Carcinogenesis ◽

Primary Human Keratinocytes ◽

Tlr9 Expression ◽

Hpv Types ◽

E6 And E7 Oncoproteins ◽

E6 And E7

ABSTRACT Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis. IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.

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Trichostatin A Up-Regulates Human Papillomavirus Type 11 Upstream Regulatory Region-E6 Promoter Activity in Undifferentiated Primary Human Keratinocytes

Journal of Virology ◽

10.1128/jvi.73.6.5026-5033.1999 ◽

1999 ◽

Vol 73 (6) ◽

pp. 5026-5033 ◽

Cited By ~ 30

Author(s):

Wei Zhao ◽

Francisco Noya ◽

Wen Yong Chen ◽

Tim M. Townes ◽

Louise T. Chow ◽

...

Keyword(s):

Human Papillomavirus ◽

Reporter Gene ◽

Trichostatin A ◽

Regulatory Region ◽

Specific Inhibitor ◽

Human Keratinocytes ◽

Upstream Regulatory Region ◽

Basal Cells ◽

Primary Human Keratinocytes ◽

Submerged Cultures

ABSTRACT Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using thelacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin.

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Cryptotanshinone from Salvia miltiorrhiza Bunge lowers the Cytokeratin 1/10 expression in primary human Keratinocytes in vitro

10.1055/s-0037-1608052 ◽

2017 ◽

Author(s):

S Esch ◽

A Hensel ◽

S Brandt ◽

S König

Keyword(s):

Salvia Miltiorrhiza ◽

Human Keratinocytes ◽

Salvia Miltiorrhiza Bunge ◽

Primary Human Keratinocytes

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Faculty Opinions recommendation of Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1144927.602030 ◽

2009 ◽

Author(s):

Cheng-Ming Chiang

Keyword(s):

Squamous Epithelium ◽

Human Keratinocytes ◽

Primary Human Keratinocytes

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Novel mutation in the TGFBI gene in a Moroccan family with atypical corneal dystrophy: a case report

BMC Medical Genomics ◽

10.1186/s12920-020-00861-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yahya Benbouchta ◽

Imane Cherkaoui Jaouad ◽

Habiba Tazi ◽

Hamza Elorch ◽

Mouna Ouhenach ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Clinical Symptoms ◽

Age Of Onset ◽

Novel Mutation ◽

Corneal Dystrophy ◽

Heterozygous Mutation ◽

Large Variability ◽

Whole Exome ◽

Moroccan Family

Abstract Background Corneal dystrophies (CDs) are a heterogeneous group of bilateral, genetically determined, noninflammatory bilateral corneal diseases that are usually limited to the cornea. CD is characterized by a large variability in the age of onset, evolution and visual impact and the accumulation of insoluble deposits at different depths in the cornea. Clinical symptoms revealed bilateral multiple superficial, epithelial, and stromal anterior granular opacities in different stages of severity among three patients of this family. A total of 99 genes are involved in CDs. The aim of this study was to identify pathogenic variants causing atypical corneal dystrophy in a large Moroccan family and to describe the clinical phenotype with severely different stages of evolution. Case presentation In this study, we report a large Moroccan family with CD. Whole-exome sequencing (WES) was performed in the three affected members who shared a phenotype of corneal dystrophy in different stages of severity. Variant validation and familial segregation were performed by Sanger sequencing in affected sisters and mothers and in two unaffected brothers. Whole-exome sequencing showed a novel heterozygous mutation (c.1772C > A; p.Ser591Tyr) in the TGFBI gene. Clinical examinations demonstrated bilaterally multiple superficial, epithelial and stromal anterior granular opacities in different stages of severity among three patients in this family. Conclusions This report describes a novel mutation in the TGFBI gene found in three family members affected by different phenotypic aspects. This mutation is associated with Thiel-Behnke corneal dystrophy; therefore, it could be considered a novel phenotype genotype correlation, which will help in genetic counselling for this family.

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Identification of a novel mutation of RUNX2 in a family with supernumerary teeth and craniofacial dysplasia by whole-exome sequencing

Medicine ◽

10.1097/md.0000000000011328 ◽

2018 ◽

Vol 97 (32) ◽

pp. e11328 ◽

Cited By ~ 7

Author(s):

Dan Ma ◽

Xuxia Wang ◽

Jun Guo ◽

Jun Zhang ◽

Tao Cai

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Novel Mutation ◽

Supernumerary Teeth ◽

Whole Exome

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Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

International Journal of Molecular Sciences ◽

10.3390/ijms22115538 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5538

Author(s):

Stefania Zampieri ◽

Silvia Cattarossi ◽

Eleonora Pavan ◽

Antonio Barbato ◽

Agata Fiumara ◽

...

Keyword(s):

Exome Sequencing ◽

Molecular Diagnosis ◽

Gaucher Disease ◽

Novel Mutation ◽

Large Deletion ◽

Clinical Exome Sequencing ◽

Number Variation ◽

Long Range Pcr ◽

Set Up ◽

Beta Glucosidase

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

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