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.

Figure 1.

Characterization of the SPRR1B gene promoter and the effects of IL-1β and IFN-γ on promoter activity. (A) Schematic representation of 3 kb SPRR1B gene 5′-flanking region. Bent arrow: transcription start site. Several putative transcription factor-binding sites were mapped on this fragment. (B) Cloning, cell transfection, and luciferase assay results. The promoter activity of sequentially deleted SPRR1B promoter/reporter constructs is shown. For each assay, 0.4 μg SPRR1B construct and 2 ng Renilla luciferase plasmid (an internal control) were transiently cotransfected into HCE cells. SPRR1B promoter activity was assessed 48 hours after transfection and expressed as fold induction of relative luciferase activity (RLU) compared with promoterless vector. SPRR1B promoter activity in response to IL-1β or IFN-γ (20 ng/mL) was assessed 12 hours after the addition of cytokine (24 hours after transfection). Cells exposed to basal medium in the absence of cytokine were used as a control. Data are expressed as mean ± SE RLU. *P < 0.01, significant induction versus basal medium control.

Figure 2.

Characterization of transcription factor binding elements by mutagenesis and luciferase assay. (A) HCE cells were transfected with wild-type (WT) SPRR1B promoter (−620/+12), two mutant variants (mutated at ZEB1 site and CREB site, respectively), and the −200/+12 construct. Relative luciferase activity (RLU), expressed as fold induction relative to promoterless vector, were measured in the presence and absence of IL-1β and IFN-γ. Data are expressed as mean ± SE RLU. *P < 0.01, significant induction versus basal medium control. (B) CREB and ZEB1 transcription factor-binding elements are underlined. Mutated base pairs are highlighted using boldface type.

Figure 3.

Competitive EMSA analysis of transcription factors on the SPRR1B promoter that mediate the stimulatory effects of IL-1β and IFN-γ. A digoxigenin-labeled, double stranded-DNA probe (60 fM) was added to 5 μg nuclear extract (NE) in protein-DNA binding buffer. Protein-DNA complexes were separated using a gradient (4%–20%) TBE gel, transferred to a nylon membrane, and detected by chemiluminescence. Unlabeled double-stranded DNA was used as competitor at 125×. Lane 1, no NE; lane 2, NE alone; lanes 3–6, cytokine-stimulated NE; lane 4, mutant (mut) probe was added; lane 5, wild-type (wt) competitor was added; lane 6, mutant competitor was added. (A) Dig-labeled probe containing CREB-binding element in the presence and absence of IL-1β. (B) Dig-labeled probe containing ZEB1-binding element in the presence and absence of IFN-γ. Images are representative of three independent experiments. NS, nonspecific binding.

Figure 4.

The involvement of MAPK signaling in IL-1β– and IFN-γ–induced SPRR1B promoter activity. HCE cells transfected with SPRR1B −620bp/luciferase reporter construct were exposed to IL-1β or IFN-γ in the presence and absence of chemical inhibitors directed against (A) JNK (SP60012, 10 μM), (B) ERK (PD98059, 25 μM), and (C) p38 (SB202190, 10 μM). Data represent the fold-induction of relative luciferase activity (mean ± SE). *P < 0.01, significant induction.

Figure 5.

Effect of the p38 MAPK inhibitor SB202190 on SPRR1B gene expression in response to IL-1β and IFN-γ. Endogenous SPRR1B mRNA was examined by real-time RT-PCR using gene expression assay. Total RNA was isolated from HCE cells pretreated for 45 minutes with DMSO (vehicle) or p38 inhibitor (10 μM), followed by 12-hour exposure to IL-1β (20 ng/μL) or IFN-γ (20 ng/μL), either alone or in combination with the inhibitor. mRNA expression level data represented as fold-induction (mean ± SE) compared with 1-fold defined by DMSO-treated control. *P < 0.01, significant induction.

Figure 6.

Effects of DNM p38α and p38β2 on SPRR1B gene expression. (A) Effect of DNM p38 (α and β2) on SPRR1B promoter activity was examined by dual luciferase assay. HCE cells were cotransfected with the SPRR1B promoter construct (620 bp) and with DNM p38α, DNM p38β2, or empty vector control. Relative luciferase activity of the SPRR1B promoter was examined in the presence and absence of IL-1β and IFN-γ (20 ng/mL). Data are expressed as mean ± SE RLU. *P < 0.01, significant induction. (B) Endogenous SPRR1B mRNA was examined by real-time RT-PCR using gene expression assays. HCE cells were transfected with p38α or β2 DNM or empty vector control. Basal medium with and without IL-1β or IFN-γ (20 ng/mL) was added to HCE cells 36 hours after transfection and was maintained for 12 hours. Fold change in SPRR1B mRNA expression in the presence and absence of DNM was determined compared with control. *P < 0.01, significant induction.

Figure 7.

p38-Dependent regulation of SPRR1B mRNA and protein expression in primary human corneolimbal epithelial cells. Primary cells were pretreated with p38 inhibitor SB202190 (10 μM) or vehicle (DMSO) alone in KSFM for 45 minutes, followed by 6- and 24-hour exposures to IL-1β (20 ng/mL) alone or in combination with the inhibitor for mRNA and protein studies, respectively. (A) SPRR1B mRNA was examined by quantitative real-time PCR using gene expression assay (as in Fig. 5). With the ΔΔC_T method, SPRR1B mRNA expression was normalized using endogenous GAPDH and is shown as relative fold increase (using control as 1-fold). SPRR1B transcription was upregulated 1.77-fold compared with control (DMSO) in response to IL-1β. *P < 0.05, significant induction. Data are representative of three independent experiments using six corneolimbal rims. (B) SPRR1B protein was visualized by immunofluorescence. Cytoplasmic SPRR1B (red) was well defined in clusters of cells stimulated by IL-1β cytokine in the absence of SB202190 (IL-1β+/SB202190−) but not in the presence of SB202190 (IL-1β+/SB202190+). Scale bar, 10 μm.

Figure 8.

Model depicting cytokine regulation of squamous metaplasia biomarker SPRR1B expression in human corneal epithelial cells. Ligation of cytokines IL-1β and IFN-γ to their cell surface receptors led to the activation of p38α/β2 MAPKs. Downstream of p38, IL-1β and IFN-γ signals recruit their individual downstream transcription factors to activate CREB- and ZEB1-response elements on the SPRR1B promoter, respectively.