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Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes.

ABSTRACT: Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.

SUBMITTER: Darbro BW

PROVIDER: S-EPMC2020850 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes.

Darbro Benjamin W BW Schneider Galen B GB Klingelhutz Aloysius J AJ

The Journal of investigative dermatology 20050901 3

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under th ...[more]

PMID: 16117791

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Project description:This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1–deficient (Bmi-1–/–), Bmi-1 and p16INK4a double-knockout (Bmi-1–/–p16INK4a–/–), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)–dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1–/– and Bmi-1–/–p16INK4a–/– mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1–/– mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α–dependent barrier permeability and aging. Accumulated p16INK4a combined with occludin at the 1st–160th residue in cytoplasm of intestinal epithelium cells from Bmi-1–/– mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16INK4a deletion could maintain barrier function and microbiota balance in Bmi-1–/– mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16INK4a accumulation. The clearance of p16INK4a-positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1–160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16INK4a with occludin for protecting TJ.

Dataset Information

Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes.

Publications

Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes.

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