Dysfunction of the stress-responsive FOXC1 transcription factor contributes to the earlier-onset glaucoma observed in Axenfeld-Rieger syndrome patients

Abstract

Mutations in the Forkhead Box C1 (FOXC1) transcription factor gene are associated with Axenfeld-Rieger syndrome (ARS), a developmental disorder affecting structures in the anterior segment of the eye. Approximately 75% of ARS patients with FOXC1 mutations develop earlier-onset glaucoma. Constant exposure of the trabecular meshwork (TM), located in the anterior segment of the eye, to oxidative stress is predicted to be a risk factor for developing glaucoma. Stress-induced death of TM cells results in dysfunction of the TM, leading to elevated intraocular pressure, which is a major risk factor for developing glaucoma. FOXC1 is predicted to maintain homeostasis in TM cells by regulating genes that are important for stress response. In this study, we show that a member of the heat-shock 70 family of proteins, HSPA6, is a target gene of FOXC1. HSPA6 protein, which is only induced under severe oxidative stress conditions, has a protective function in human trabecular meshwork (HTM) cells. We also show that FOXC1 is anti-apoptotic as knocking down FOXC1 significantly decreases HTM cell viability. In addition, we show that FOXC1 itself responds to stress as exposure of cells to H2O2-induced oxidative stress reduces FOXC1 levels and activity. Conditions that decrease FOXC1 function, such as exposure of cells to oxidative stress and FOXC1 ARS mutations, compromise the ability of TM cells to effectively respond to environmental stresses. Dysfunction of FOXC1 contributes to the death of TM cells, an important step in the development of glaucoma.

Figure 1

HSPA6 is a target gene of FOXC1 in HTM cells. HTM cells were transfected with FOXC1 siRNA or control siRNA. After transfection, RNA was harvested and subjected to (a) northern blot analysis or (b) qPCR analysis. (a) A HSPA6 probe was radiolabeled and hybridized to the blot. Relative to control siRNA, HSPA6 RNA levels significantly decreased when FOXC1 was knocked down (P=0.01, n=4, Student's t-test). (b) qPCR results show that relative to control siRNA, HSPA6 RNA levels significantly decreased after FOXC1 was knocked down (*P<0.01, n=4, Student's t-test)

Figure 2

FOX-binding sites are present in the HSPA6 upstream region. Chromatin immunoprecipitation (ChIP) assay identified BS1, located ∼4800 bp upstream of the HSPA6 transcription start site, as a bona fide FOXC1-binding site. The BS3 region containing two FOX consensus binding sites located ∼1300 bp upstream of the HSPA6 transcription site was also able to bind FOXC1. HTM cells were crosslinked and immunoprecipitated against FOXC1. Acetylated histone 3 at lysine 9 (Ack9) and IgG were used as positive and negative controls for the ChIP technique, respectively. The immunoprecipitated DNA was amplified by PCR using primers flanking the potential FOXC1-binding site, identified by Possum software. Genomic DNA and water are positive and negative controls, respectively, for the PCR. Primer flanking BS2 did not amplify a product in ChIP products incubated with FOXC1 antibody, indicating that BS2 does not bind to FOXC1

Figure 3

HSPA6 protein is present only after exposure to a severe dose of H₂O₂. (a) Protein lysates were resolved on a 10% SDS-PAGE and immunoblotted using antibodies against HSPA6 or ERK1/2. Immunoblot analysis shows the presence of HSPA6 protein only in HTM cells exposed to a severe dose of H₂O₂. (b) For overexpression or knockdown of FOXC1 or HSPA6, HTM cells were first transfected with either siRNA against the gene of interest or a plasmid construct, respectively. Approximately 24 h post transfection, HTM cells were exposed to a low dose (500 μM), high dose (1000 μM), or severe dose (500 μM followed by 1000 μM) of H₂O₂

Figure 4

FOXC1 knockdown increases HSPA6 levels after exposure to a severe dose of H₂O₂. (a) Protein lysates were resolved on a 10% SDS-PAGE and immunoblotted using antibodies against HSPA6 or ERK1/2. Knocking down FOXC1 in severely oxidatively stressed cells resulted in a significant increase (*) in HSPA6 protein (P=0.02. Student's t-test). (b) Total RNA was extracted and HSPA6 and HPRT1 RNA levels were quantified by qPCR. HSPA6 RNA levels were normalized to HPRT1 RNA levels. qPCR results show that HSPA6 RNA levels increase after exposure to a severe dose of H₂O₂, further confirming that HSPA6 is a target gene of FOXC1. Quantification is based on triplicated experiments

Figure 5

Knocking down HSPA6 increases apoptotic cell death after exposure of HTM cells to ‘severe' H₂O₂-induced oxidative stress. (a) Cell viability was quantified by staining with trypan blue. HSPA6 knockdown resulted in a significant decrease (*) in cell viability in cells exposed to a ‘severe dose' of H₂O₂. (b) Immunoblot analysis showed that decreasing HSPA6 significantly increased (*) cleaved PARP-1 levels after cells were exposed to a ‘severe dose' of H₂O₂. Quantification is based on triplicated experiments

Figure 6

Knocking down FOXC1 increases apoptotic cell death after exposure of HTM cells to ‘low dose' and ‘high dose' H₂O₂-induced oxidative stress. (a) Cell viability was quantified by staining with trypan blue. Knocking down FOXC1 significantly decreased (*) cell viability in cells exposed to a ‘low dose', ‘high dose', and ‘severe dose' of H₂O₂. Immunoblot analysis showed that (*) decreasing FOXC1 significantly increased the apoptotic markers (b) cleaved PARP-1 and (c) cleaved caspase-7 after cells were exposed to a ‘low dose' and/or ‘high dose' of H₂O₂. FOXC1 knockdown had no effect on cell viability or the apoptotic markers when cells were exposed to a ‘severe dose' of H₂O₂. Quantification is based on triplicated experiments

Figure 7

Overexpression of wild-type and mutant FOXC1s has differential effects on cell viability. HTM cells were transfected with (a) wild-type FOXC1 or (b) three mutant FOXC1s; L130F, W152G, and S131L. Transfected HTM cells were subjected to a low dose (500 μM), high dose (1000 μM), or severe dose (500 μM followed by 1000 μM) of H₂O₂. Cell viability was quantified by staining with trypan blue. Wild-type FOXC1 overexpression significantly increased (*) viability in cells exposed to a ‘high dose' of H₂O₂ (P<0.01, Student's t-test). Under severe dose conditions, there was no significant difference between wild-type FOXC1 and the L130F, W152G, and S131L mutants with P-values of 0.06, 0.20, and 0.13 respectively. Mutant FOXC1 overexpression significantly decreased (*) viability in cells (P<0.01, Student's t-test). (c) Immunoblot analysis showed that FOXC1 protein levels decreased when HTM cells were exposed to H₂O₂-induced oxidative stress. Quantification is based on triplicated experiments

Figure 8

H₂O₂-induced oxidative stress decreases FOXC1 levels. (a) Nuclear extracts were resolved by 10% SDS-PAGE and immunoblotted using antibodies against endogenous FOXC1 and TFIID. (b) Total RNA was extracted and FOXC1 and HPRT1 RNA levels were quantified by qPCR. FOXC1 RNA levels were normalized to HPRT1 RNA levels. The * indicates a P value <0.01 (Student's t-test) relative to ‘no H₂O₂' samples. (c) FOXC1 was able to transactivate the luciferase reporter gene under ‘no H₂O₂' conditions. The transactivation ability of FOXC1 significantly decreased (*) in HeLa cells treated with a ‘low dose' and ‘severe dose' of H₂O₂. (d) Immunoblot analysis showed that exogenous FOXC1 protein levels decreased when cells were treated with H₂O₂. Lysates were resolved by 10% SDS-PAGE and immunoblotted using Xpress and tubulin antibodies. Quantification is based on triplicated experiments