Molecular mechanism of proinflammatory cytokine-mediated squamous metaplasia in human corneal epithelial cells.
OBJECTIVE
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.
Li
Shimin
S
Francis I. Proctor Foundation, University of California, San Francisco, CA 94143-0412, USA.
Gallup
Marianne
M
Chen
Ying-Ting
YT
McNamara
Nancy A
NA
D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D003315:Cornea / Q000473:pathology; D055233:Cornified Envelope Proline-Rich Proteins / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008679:Metaplasia; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071799:Zinc Finger E-box-Binding Homeobox 1; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D055233:Cornified Envelope Proline-Rich Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D018398:Homeodomain Proteins; D053583:Interleukin-1beta; D012333:RNA, Messenger; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; C524533:SPRR1B protein, human; D007371:Interferon-gamma; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4