Molecular mechanism of proinflammatory cytokine-mediated squamous metaplasia in human corneal epithelial cells.

Abstract

PURPOSE:

The cornified envelope protein small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia. Proinflammatory cytokines IL-1beta and IFN-gamma are potent inducers of ocular surface keratinization and SPRR1B expression. Here the molecular mechanisms controlling SPRR1B gene expression in response to IL-1beta and IFN-gamma are elucidated.

METHODS:

A 3-kb fragment of the SPRR1B gene 5'-flanking region was amplified from human chromosome 1, sequentially deleted, and cloned into a luciferase vector. Constructs were transiently transfected into human corneal epithelial cells, and activity was assessed in response to IL-1beta, IFN-gamma, or basal medium. Functional cis-elements responding to IL-1beta and IFN-gamma were characterized by site-directed mutagenesis and gel mobility shift assay. Effects of mitogen-activated protein kinases p38, ERK, and JNK were assessed using inhibitors and dominant-negative mutants. Results were validated by real-time RT-PCR.

RESULTS:

The first 620 bp of the SPRR1B 5'-flanking region regulated constitutive expression and increased promoter activity in response to IL-1beta and IFN-gamma. Corresponding cis-elements for IL-1beta and IFN-gamma were bound by cAMP response element binding protein (CREB) and zinc-finger E-box binding homeobox 1 (ZEB1), respectively. Inhibition of p38 abolished the stimulatory effects of IL-1beta and IFN-gamma on SPRR1B, whereas inhibition of JNK and ERK had no effect. Dominant-negative mutants targeting p38alpha and p38beta2 blocked cytokine-induced SPRR1B promoter activity and mRNA expression.

CONCLUSIONS:

SPRR1B is upregulated by the proinflammatory cytokines IL-1beta and IFN-gamma via p38 MAPK-mediated signaling pathways that lead to the activation of transcription factors CREB and ZEB1, respectively. These results identify key intracellular signaling intermediates involved in the pathogenesis of immune-mediated ocular surface squamous metaplasia.

• Authors: Shimin Li, Marianne Gallup, Ying-Ting Chen, Nancy A McNamara
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 51 Issue 5 Pg. 2466-75 (May 2010) ISSN: 1552-5783 [Electronic] United States
• PMID: 20042643 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Antigens, Polyomavirus Transforming
• Biomarkers
• Cornified Envelope Proline-Rich Proteins
• Cyclic AMP Response Element-Binding Protein
• Homeodomain Proteins
• Interleukin-1beta
• RNA, Messenger
• Transcription Factors
• ZEB1 protein, human
• Zinc Finger E-box-Binding Homeobox 1
• SPRR1B protein, human
• Interferon-gamma
• Extracellular Signal-Regulated MAP Kinases
• p38 Mitogen-Activated Protein Kinases
• MAP Kinase Kinase 4

Topics

• Antigens, Polyomavirus Transforming
• Biomarkers
• Cell Culture Techniques
• Cell Line, Transformed
• Cornea (pathology)
• Cornified Envelope Proline-Rich Proteins (genetics)
• Cyclic AMP Response Element-Binding Protein (genetics, metabolism)
• Electrophoretic Mobility Shift Assay
• Epithelium, Corneal (drug effects, metabolism, pathology)
• Extracellular Signal-Regulated MAP Kinases (metabolism)
• Gene Expression Regulation (physiology)
• Homeodomain Proteins (genetics, metabolism)
• Humans
• Interferon-gamma (pharmacology)
• Interleukin-1beta (pharmacology)
• MAP Kinase Kinase 4 (metabolism)
• Metaplasia
• Mutagenesis, Site-Directed
• RNA, Messenger (metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Transcription Factors (genetics, metabolism)
• Transfection
• Zinc Finger E-box-Binding Homeobox 1
• p38 Mitogen-Activated Protein Kinases (metabolism)